Colchicine compositions and methods

ABSTRACT

Stable ultrapure colchicine compositions comprising ultrapure colchicine and a pharmaceutically acceptable excipient are described. The compositions can be tablets. Methods for preparing such compositions and methods of use are also disclosed. Methods of treating gout flares with colchicine compositions are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/175,062, filed Jul. 1, 2011; which is a continuation of U.S.application Ser. No. 12/687,406, filed Jan. 14, 2010, now issued as U.S.Pat. No. 7,981,938; which is a continuation of U.S. application Ser. No.12/545,377, filed Aug. 21, 2009, now abandoned; which is a continuationof U.S. application Ser. No. 12/465,210, filed May 13, 2009, nowabandoned, and a continuation of U.S. application Ser. No. 12/407,980,filed Mar. 20, 2009, now issued as U.S. Pat. No. 7,964,647; which is acontinuation of U.S. application Ser. No. 12/246,034, filed Oct. 6,2008, now abandoned; which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/977,796 filed Oct. 5, 2007 and U.S. ProvisionalApplication Ser. No. 61/090,965 filed Aug. 22, 2008; each of theabove-named applications is hereby incorporated by reference in itsentirety.

BACKGROUND

This application relates to colchicine compositions for therapeuticpurposes, specifically ultrapure colchicine, and methods of making andusing the colchicine compositions.

Colchicine, chemical name(−)-N-[(7S,12a5)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]-acetamide,is a pale yellow powder soluble in water in 1:25 dilution.

Colchicine is an alkaloid found in extracts of certain plants such asColchicum autumnale and Gloriosa superba. Colchicine arrests celldivision in animals and plants. It has adversely affectedspermatogenesis in humans and in some animal species under certainconditions.

Gout (or gouty arthritis) is a disease caused by a build up of uric aciddue to an overproduction of uric acid or a reduced ability of the kidneyto get rid of uric acid. It is more common in males, postmenopausalwomen, and people with high blood pressure. Heavy alcohol use, diabetes,obesity, sickle cell anemia, and kidney disease also increase the risk.The condition may also develop in people who take drugs that interferewith uric acid excretion.

In gout, monosodium urate or uric acid crystals are deposited on thearticular cartilage of joints, tendons and surrounding tissues due toelevated concentrations of uric acid in the blood stream. This provokesan inflammatory reaction of these tissues. Gout is characterized byexcruciating, sudden, unexpected, burning pain, as well as swelling,redness, warmness, and stiffness in the affected joint. Low-grade fevermay also be present. The patient usually suffers from two sources ofpain. The crystals inside the joint cause intense pain whenever theaffected area is moved. The inflammation of the tissues around the jointalso causes the skin to be swollen, tender and sore if it is evenslightly touched. For example, a blanket or even the lightest sheetdraping over the affected area could cause extreme pain.

Acute gouty arthritis (alternatively referred to as a gout flare or agout attack) is a sudden attack of pain in affected joints, especiallyin the feet and legs. Chronic gout involves repeated attacks of jointpain.

In acute gouty arthritis, symptoms develop suddenly and usually involveonly one or a few joints. The big toe, knee, or ankle joints are mostoften affected. The pain frequently starts during the night and is oftendescribed as throbbing, crushing, or excruciating. The joint appearsinfected with signs of warmth, redness, and tenderness. The attacks ofpainful joints may go away in several days, but may return from time totime. Subsequent attacks usually last longer. Some people may progressto chronic gout (chronic gouty arthritis), while others may have nofurther attacks.

If several attacks of gout occur each year, it can lead to jointdeformity and limited motion in joints. Uric acid deposits, calledtophi, develop in cartilage tissue, tendons, and soft tissues. Thesetophi usually develop only after a patient has suffered from the diseasefor many years. Deposits also can occur in the kidneys, leading tochronic kidney failure.

Colchicine can be used for treating adults with acute gouty arthritisand pain in attacks of acute gouty arthritis, and also can be usedbeneficially for treating adults with chronic gout for prophylaxis ofacute gout flares. Although its exact mode of action in the relief ofgout is not completely understood, colchicine is known to decrease theinflammatory response to urate crystal deposition by inhibitingmigration of leukocytes, to interfere with urate deposition bydecreasing lactic acid production by leukocytes, to interfere with kininformation and to diminish phagocytosis and the subsequentanti-inflammatory response. The anti-inflammatory effect of colchicineis relatively selective for acute gouty arthritis. However, other typesof arthritis occasionally respond. It is neither an analgesic nor auricosuric and will not prevent progression to chronic gouty arthritis.It does have a prophylactic, suppressive effect that helps to reduce theincidence of acute attacks and to relieve the residual pain and milddiscomfort that patients with gout occasionally experience. In someinstances, non-steroidal anti-inflammatory drugs (NSAIDs) may also beprescribed to relieve pain and inflammation in acute gouty arthritisattacks. Strong painkillers, such as codeine, or corticosteroids mayalso be prescribed to relieve the pain.

Colchicine is rapidly absorbed from the gastrointestinal tract. Peakconcentrations occur in 0.5 to 2 hours. The drug and its metabolites aredistributed in leukocytes, kidneys, liver, spleen and the intestinaltract. Colchicine is metabolized in the liver and excreted primarily inthe feces with 10 to 20% eliminated unchanged in the urine.

There remains a need for pure forms of colchicine having low levels ofimpurities for pharmaceutical use to minimize the potential for sideeffects in patients taking colchicine pharmaceutical products and tominimize the need for costly toxicity testing required for approval ofpharmaceutical products comprising conventional colchicine having highlevels of individual or total impurities. In particular, there is a needfor stable compositions comprising ultrapure colchicine.

SUMMARY

Disclosed herein are colchicine compositions.

In one embodiment, the colchicine composition comprises ultrapurecolchicine, wherein the ultrapure colchicine comprises no more thanabout 3.0% total impurities, specifically no more than about 2.0% totalimpurities, and a pharmaceutically acceptable excipient.

In another embodiment, the colchicine composition comprises ultrapurecolchicine, wherein the ultrapure colchicine comprises no more than 3.0%total impurities, specifically no more than about 2.0% total impurities,a filler, a binder, and a disintegrant.

In yet another embodiment, the colchicine composition comprises about0.6 mgA colchicine, about 12 to about 16 mg pregelatinized starch, about20 to about 24 mg microcrystalline cellulose, about 3.9 to about 4.7 mgsodium starch glycolate, about 0.5 to about 0.7 mg magnesium stearate,and an amount of lactose monohydrate such that the colchicine dosageform has a total weight of about 100 mg.

In an embodiment, the colchicine composition comprises colchicine; and apharmaceutically acceptable excipient; wherein the colchicinecomposition comprises no more than about 3.5% total impurities and nomore than 0.42% N-deacetyl-N-formyl colchicine.

In an embodiment, the colchicine composition comprises colchicine; and apharmaceutically acceptable excipient, wherein the colchicinecomposition has 0.6 mgA colchicine, wherein a single dose of the 0.6 mgAcolchicine composition has enhanced bioavailability as compared to asingle dose of a pharmaceutical product comprising 0.5 mg colchicineafter potency correction for colchicine.

In an embodiment, the colchicine composition comprises colchicine; and apharmaceutically acceptable excipient, wherein the colchicinecomposition has equivalent bioavailability when administered in a fed ora fasted state.

In an embodiment, the colchicine composition comprises colchicine; and apharmaceutically acceptable excipient, wherein administration of asingle dose of the colchicine composition to a human provides a Cmaxbetween about 1.3 ng/mL and about 4.0 ng/mL, an AUC_(0-t) between about4.4 ng-hr/mL and about 30.8 ng-hr/mL, or an AUC_(0-INF) between about6.7 ng-hr/mL and about 27.8 ng-hr/mL.

Methods of making the colchicine compositions are also disclosed herein.

In an embodiment, the method comprises wet granulating ultrapurecolchicine, wherein the ultrapure colchicine comprises no more thanabout 3.0% of total impurities, with a pharmaceutically acceptableexcipient to obtain wet granules, and mixing the granules with adisintegrant to obtain the composition.

In yet another embodiment, the method comprises wet granulatingcolchicine with a pharmaceutically acceptable excipient to obtain wetgranules; drying the wet granules to obtain dried granules; milling thedried granules to obtain milled granules; mixing the milled granuleswith a disintegrant to obtain the composition; mixing the compositionwith a lubricant to obtain a tableting blend; and compressing thetableting blend to obtain a colchicine tablet.

Also disclosed herein are methods of making ultrapure colchicine.

In an embodiment, the method comprises subjecting colchicine comprisingmore than about 3.0% total impurities to column chromatography to obtaina colchicine concentrate, distilling the colchicine concentrate toobtain a colchicine distillate, and crystallizing ultrapure colchicinefrom the colchicine distillate; wherein the ultrapure colchicinecomprises no more than about 3.0% total impurities.

In another embodiment, the method comprises subjecting colchicinecomprising more than about 3.0% total impurities to neutral aluminacolumn chromatography to obtain a colchicine concentrate; distilling thecolchicine concentrate with ethyl acetate to obtain a colchicinedistillate; and crystallizing ultrapure colchicine from the colchicinedistillate in ethyl acetate; wherein the ultrapure colchicine comprisesno more than about 3.0% total impurities.

In still another embodiment, the method comprises subjecting colchicinecomprising more than about 3.0% total impurities to neutral aluminacolumn chromatography to obtain a colchicine concentrate; distilling thecolchicine concentrate with ethyl acetate to obtain a colchicinedistillate; crystallizing a purified colchicine from the colchicinedistillate in ethyl acetate; washing the purified colchicine with ethylacetate to obtain a washed purified colchicine; and drying the washedpurified colchicine to obtain ultrapure colchicine, wherein theultrapure colchicine comprises no more than about 3.0% total impurities.

Also disclosed are methods of treating a patient with the colchicinecompositions.

In an embodiment, the method comprises administering a dosing regimen toa patient having an acute gouty arthritis attack, wherein the dosingregimen consists of two colchicine dosage forms at the onset of theacute gouty arthritis attack, followed by one colchicine dosage form inabout one hour, wherein a colchicine dosage form comprises about 0.6 mgAcolchicine.

In an embodiment, the method comprises administering less than about 2mg of colchicine to a patient over a period of about one hour, whereinthe patient is experiencing an acute gouty arthritis attack.

In an embodiment, the method comprises administering to a humanexperiencing an acute gouty arthritis attack a colchicine composition,wherein the composition is effective to provide a colchicine plasmaconcentration profile having an area under the plasma colchicineconcentration curve from time 0 to infinity (AUC_(0-INF)) of about 34.2ng-hr/mL to about 74.1 ng-hr/mL, an area under the plasma colchicineconcentration curve from time 0 to time t (AUC_(0-t)) of about 28.8ng-hr/mL to about 58.9 ng-hr/mL, or a maximum plasma colchicineconcentration (Cmax) of about 3.2 ng/mL to about 11.4 ng/mL for amaximum total dose of about 1.8 mg colchicine.

In an embodiment, the method comprises administering a dosing regimen toa patient having an acute gouty arthritis attack, wherein the dosingregimen consists of two colchicine dosage forms at the onset of theacute gouty arthritis attack, followed by one colchicine dosage form inabout one hour, wherein a colchicine dosage form comprises about 0.6 mgAcolchicine, wherein the dosing regimen is effective to provide an areaunder the plasma colchicine concentration curve from time 0 to infinity(AUC_(0-INF)) of about 34.2 ng-hr/mL to about 74.1 ng-hr/mL, an areaunder the plasma colchicine concentration curve from time 0 to time t(AUC_(0-t)) of about 28.8 ng-hr/mL to about 58.9 ng-hr/mL, or a maximumplasma colchicine concentration (Cmax) of about 3.2 ng/mL to about 11.4ng/mL.

In an embodiment, the method comprises administering a dosing regimen toa patient having an acute gouty arthritis attack, wherein the dosingregimen consists of two colchicine dosage forms at the onset of theacute gouty arthritis attack, followed by one colchicine dosage form inabout one hour, wherein a colchicine dosage form comprises about 0.6 mgAcolchicine, wherein the dosing regimen is effective to provide acolchicine plasma concentration profile which has a maximum plasmacolchicine concentration (Cmax) which is at least 80% of plasma Cmaxprovided by a dosing regimen of two dosage forms, followed by one dosageform about every hour later for 6 hours.

In an embodiment, the method comprises administering a dosing regimen toa patient having an acute gouty arthritis attack, wherein the dosingregimen consists of two colchicine dosage forms at the onset of theacute gouty arthritis attack, followed by one colchicine dosage form inabout one hour, wherein a colchicine dosage form comprises about 0.6 mgAcolchicine, wherein the odds of a patient being a responder to thedosing regimen are not statistically different from the odds of being aresponder to a second dosing regimen consisting of two colchicine dosageforms at the onset of the acute gouty arthritis attack, followed by onecolchicine dosage form every hour for 6 hours, wherein a responder is apatient obtaining a ≧50% improvement in pain at 24 hours after the firstdose, without taking an additional active agent for reducing pain of theacute gouty arthritis attack.

In an embodiment, the method comprises administering a dosing regimen toa patient having an acute gouty arthritis attack, wherein the dosingregimen consists of two colchicine dosage forms at the onset of theacute gouty arthritis attack, followed by one colchicine dosage form inabout one hour, wherein a colchicine dosage form comprises about 0.6 mgAcolchicine, wherein in a randomized, placebo-controlled study of thedosing regimen in patients with an acute gouty arthritis attack, thefraction of patients that experienced a given % improvement in pain at24 hrs after first dose is shown in FIG. 1.

These and other embodiments, advantages and features of the presentinvention become clear when detailed description and examples areprovided in subsequent sections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fraction of all patients improved at 24 hrs post-firstdose of colchicine, regardless of pain rescue, as a function of thepercent improvement in pain determined in the study of Example 3.

DETAILED DESCRIPTION

Disclosed herein are compositions comprising ultrapure colchicine and apharmaceutically acceptable excipient. Herein, “ultrapure colchicine”means colchicine comprising no more than about 3.0% of total impurities,measured chromatographically as described below, specifically the ultrapure colchicine comprises no more than about 2.0% of total impurities,more specifically no more than about 1.0% of total impurities, or evenmore specifically no more than about 0.5% of total impurities. In someembodiments, the ultrapure colchicine comprises no more than about 0.10%of N-deacetyl-N-formyl colchicine, measured chromatographically. In someembodiments, the ultrapure colchicine is purified from a botanicalsource. Methods of making ultrapure colchicine and the compositionscomprising the ultrapure colchicine, methods of treating variousconditions using the compositions. Dosing regimens are also disclosed.

Not wishing to be bound by theory, it is postulated that the propertiesof colchicine as an antimitotic agent (e.g. its tubulin bindingproperties) or its effects on Pgp transporter properties provide thetherapeutic effects of colchicine described herein. The methodsdescribed herein therefore also contemplate the use of an antimitoticagent with at least one pharmaceutically acceptable excipient. Anantimitotic agent can be a drug that prevents or inhibits mitotis, orcell division.

In the specification and claims that follow, references will be made toa number of terms which shall be defined to have the following meaning.

The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced item. Theterm “or” means “and/or”. The terms “comprising”, “having”, “including”,and “containing” are to be construed as open-ended terms (i.e., meaning“including, but not limited to”) unless otherwise noted.

An “active agent” means a compound (including for example, colchicine),element, or mixture that when administered to a patient, alone or incombination with another compound, element, or mixture, confers,directly or indirectly, a physiological effect on the patient. Theindirect physiological effect may occur via a metabolite or otherindirect mechanism. When the active agent is a compound, then salts,solvates (including hydrates), and co-crystals of the free compound orsalt, crystalline forms, non-crystalline forms, and any polymorphs ofthe compound are contemplated herein. Compounds may contain one or moreasymmetric elements such as stereogenic centers, stereogenic axes andthe like, e.g., asymmetric carbon atoms, so that the compounds can existin different stereoisomeric forms. These compounds can be, for example,racemates or optically active forms. For compounds with two or moreasymmetric elements, these compounds can additionally be mixtures ofdiastereomers. For compounds having asymmetric centers, all opticalisomers in pure form and mixtures thereof are encompassed. In addition,compounds with carbon-carbon double bonds may occur in Z- and E-forms,with all isomeric forms of the compounds. In these situations, thesingle enantiomers, i.e., optically active forms can be obtained byasymmetric synthesis, synthesis from optically pure precursors, or byresolution of the racemates. Resolution of the racemates can also beaccomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example, a chiral HPLC column. All forms are contemplatedherein regardless of the methods used to obtain them.

“Bioavailability” means the extent or rate at which an active agent isabsorbed into a living system or is made available at the site ofphysiological activity. For active agents that are intended to beabsorbed into the bloodstream, bioavailability data for a givenformulation may provide an estimate of the relative fraction of theadministered dose that is absorbed into the systemic circulation.“Bioavailability” can be characterized by one or more pharmacokineticparameters.

“Bioequivalence” or “equivalent bioavailability” means the absence of asignificant difference in the rate or extent to which the active agentin pharmaceutical equivalents or pharmaceutical alternatives is absorbedinto a living system or is made available at the site of physiologicalactivity or the absence of a significant difference in the rate orextent to which the active agent in a pharmaceutical composition isabsorbed into a living system or is made available at the site ofphysiological activity when administered by two different methods (e.g.,dosing under non-fasted versus fasted conditions). Bioequivalence can bedetermined by comparing in vitro dissolution testing data for two dosageforms or two dosing conditions or by comparing pharmacokineticparameters for two dosage forms or two dosing conditions.

In some embodiments, two products (e.g. an inventive composition andCOL-PROBENECID®) or two methods (e.g., dosing under fed (non-fasted)versus fasted conditions) are bioequivalent if the ratio of thegeometric mean of logarithmic transformed AUC_(0-∞), AUC_(0-t), orC_(max) for the two products or two methods is about 0.80 to about 1.25;specifically if the 90% Confidence Interval (CI) limit for the ratio ofthe geometric mean of logarithmic transformed AUC_(0-∞), AUC_(0-t), orC_(max) for the two products or two methods is about 0.80 to about 1.25;more specifically if the ratios of the geometric mean of logarithmictransformed AUC_(0-∞), AUC_(0-t), and C_(max) for the two products ortwo methods are about 0.80 to about 1.25; yet more specifically if the90% Confidence Interval (CI) limits for the ratios of the geometric meanof logarithmic transformed AUC_(0-∞), AUC_(0-t), and C_(max) for the twoproducts or two methods are about 0.80 to about 1.25.

“Colchicine therapy” refers to medical treatment of a symptom, disorder,or condition by administration of colchicine. Colchicine therapy can beconsidered optimal when effective plasma levels are reached whenrequired. In addition, peak plasma values (C_(max)) should be as low aspossible so as to reduce the incidence and severity of possible sideeffects.

“Conventional colchicine” means colchicine comprising more than 3% butno more than about 5.0% total impurities, measured chromatographicallyas described below, and comprising more than about 0.10% ofN-deacetyl-N-formyl colchicine.

A “dosage form” means a unit of administration of an active agent.Examples of dosage forms include tablets, capsules, injections,suspensions, liquids, emulsions, creams, ointments, suppositories,inhalable forms, transdermal forms, and the like.

“Dosing regimen” means the dose of an active agent taken at a first timeby a patient and the interval (time or symptomatic) at which anysubsequent doses of the active agent are taken by the patient. Theadditional doses of the active agent can be different from the dosetaken at the first time.

A “dose” means the measured quantity of an active agent to be taken atone time by a patient.

“Efficacy” means the ability of an active agent administered to apatient to produce a therapeutic effect in the patient.

As used herein, the term “mgA” refers to milligrams of the activecolchicine, or the free base of colchicine, after compensating for thepotency of the batch of colchicine (i.e., after compensating forimpurities, including solvents, and salts in the colchicine). Forexample, 0.612 mg of an ultrapure colchicine free base having a totalimpurity of 2 wt % (thus a purity of 98 wt %) contains 0.6 mgA (0.612mg×0.98=0.6 mgA) of colchicine.

An “oral dosage form” means a unit dosage form for oral administration.

A “patient” means a human or non-human animal in need of medicaltreatment. Medical treatment can include treatment of an existingcondition, such as a disease or disorder, prophylactic or preventativetreatment, or diagnostic treatment. In some embodiments the patient is ahuman patient.

“Pharmaceutically acceptable” means that which is generally safe,non-toxic and neither biologically nor otherwise undesirable andincludes that which is acceptable for veterinary use as well as humanpharmaceutical use.

“Pharmaceutically acceptable salts” includes derivatives of colchicine,wherein the colchicine is modified by making acid or base addition saltsthereof, and further refers to pharmaceutically acceptable solvates,including hydrates, and co-crystals of such compounds and such salts.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid addition salts of basic residuessuch as amines; alkali or organic addition salts of acidic residues; andthe like, and combinations comprising one or more of the foregoingsalts. The pharmaceutically acceptable salts include non-toxic salts andthe quaternary ammonium salts of the colchicine. For example, non-toxicacid salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; other acceptable inorganic salts include metal salts such assodium salt, potassium salt, cesium salt, and the like; and alkalineearth metal salts, such as calcium salt, magnesium salt, and the like,and combinations comprising one or more of the foregoing salts.Pharmaceutically acceptable organic salts includes salts prepared fromorganic acids such as acetic, propionic, succinic, glycolic, stearic,lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic,esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,methanesulfonic, ethane disulfonic, oxalic, isethionic,HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like; organic amine saltssuch as triethylamine salt, pyridine salt, picoline salt, ethanolaminesalt, triethanolamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, and the like; and amino acid saltssuch as arginate, asparginate, glutamate, and the like; and combinationscomprising one or more of the foregoing salts; organic amine salts suchas triethylamine salt, pyridine salt, picoline salt, ethanolamine salt,triethanolamine salt, dicyclohexylamine salt, N,N′dibenzylethylenediamine salt, and the like; and amino acid salts such asarginate, asparginate, glutamate, and the like; and combinationscomprising one or more of the foregoing salts. All forms of suchderivatives of colchicine are contemplated herein, including allcrystalline, amorphous, and polymorph forms. Specific colchicine saltsinclude colchicine hydrochloride, colchicine dihydrochloride, andco-crystals, hydrates or solvates thereof.

“Pharmacokinetic parameters” describe the in vivo characteristics of anactive agent (or a metabolite or a surrogate marker for the activeagent) over time, such as plasma concentration (C), C_(max), C_(n), C₂₄,T_(max), and AUC. “C_(max)” is the measured plasma concentration of theactive agent at the point of maximum, or peak, concentration. “C_(min)”is the measured plasma concentration of the active agent at the point ofminimum concentration. “C_(n)” is the measured plasma concentration ofthe active agent at about n hours after administration. “C₂₄” is themeasured plasma concentration of the active agent at about 24 hoursafter administration. The term “T_(max)” refers to the time at which themeasured plasma concentration of the active agent is the highest afteradministration of the active agent. “AUC” is the area under the curve ofa graph of the measured plasma concentration of an active agent vs.time, measured from one time point to another time point. For exampleAUC_(0-t) is the area under the curve of plasma concentration versustime from time 0 to time t, where t can be the last time point withmeasurable plasma concentration for an individual formulation. TheAUC_(0-∞) or AUC_(0-INF) is the calculated area under the curve ofplasma concentration versus time from time 0 to time infinity. Insteady-state studies, AUC_(0-τ) is the area under the curve of plasmaconcentration over the dosing interval (i.e., from time 0 to time τ(tau), where tau is the length of the dosing interval. Otherpharmacokinetic parameters are the parameter K_(e) or K_(el), theterminal elimination rate constant calculated from a semi-log plot ofthe plasma concentration versus time curve; t_(1/2) the terminalelimination half-life, calculated as 0.693/K_(a); CL/F denotes theapparent total body clearance after administration, calculated as TotalDose/Total AUC_(∞); and V_(area)/F denotes the apparent total volume ofdistribution after administration, calculated as Total Dose/(TotalAUC_(∞)×K_(el)).

“Adverse event” means any untoward medical occurrence in a patientadministered an active agent and which does not necessarily have to havea causal relationship with this treatment. An adverse event (AE) cantherefore be any unfavorable and unintended sign (including an abnormallaboratory finding, for example), symptom, or disease temporallyassociated with the use of the active agent, whether or not consideredrelated to the active agent.

“Side effect” means a secondary effect resulting from taking an activeagent. The secondary effect can be a negative (unfavorable) effect(i.e., an adverse side effect) or a positive (favorable) effect.

The most frequently reported adverse side effects to colchicine therapyare gastrointestinal, specifically diarrhea; abdominal pain with cramps;nausea; and vomiting. Less frequently or rarely reported adverse sideeffects associated with colchicine therapy include anorexia,agranulocytosis, allergic dermatitis, allergic reactions, alopecia,angioedema, aplastic anemia, bone marrow depression, myopathy,neuropathy, skin rash, thrombocytopenic disorder, and urticaria.

Determining that a patient experiences an adverse side effect can beperformed by obtaining information from the patient regarding onset ofcertain symptoms which may be indicative of the adverse side effect,results of diagnostic tests indicative of the adverse side effect, andthe like.

Ultrapure colchicine comprising low levels of individual impurities ortotal impurities is highly desirable as compared to conventionallyavailable forms of colchicine. Currently, commercially available formsof colchicine often comprise high levels of impurities. Depending on thesource or the preparation process, colchicine may comprise some or allof the common impurities shown in Table 1.

TABLE 1 Common Other common Impurities Chemical Name name Impurity AN-[(7S,12aS)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9- N-deacetyl-N-tetrahydrobenzo[a]heptalen-7-yl]formamide formyl colchicine Impurity B(−)-N-[(7S,12aR)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9- Conformationaltetrahydrobenzo[a]heptalen-7-yl]-acetamide isomer Impurity CN-[(7S,7bR,10aS)-1,2,3,9-tetramethoxy-8-oxo-5,6,7,7b,8,10a-β-Lumicolchicinehexahydrobenzo[a]cyclopenta[3,4]cyclobuta[1,2-c]cyclohepten-7-yl]-acetamide Impurity DN-[(7S,12aS)-3-(β-D-glucopyranosyloxy)-1,2,10-trimethoxy-9-oxo-Colchicoside 5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]-acetamide ImpurityE N-[(7S,12aS)-3-hydroxy-1,2,10-trimethoxy-9-oxo-5,6,7,9- 3-O-demethyltetrahydrobenzo[a]heptalen-7-yl]-acetamide colchicine Impurity FN-[7S,12aS)-10-hydroxy-1,2,3-trimethoxy-9-oxo-5,6,7,9- Colchiceinetetrahydrobenzo[a]heptalen-7-yl]acetamide

In addition to the common impurities listed above, colchicine may alsocompriseN-[(7S,12aS)-2-hydroxy-1,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]-acetamide(“2-O-demethyl colchicine”) impurity. Some analytical methods cannotdifferentiate 2-O-demethyl colchicine from 3-O-demethyl colchicine.

In addition to the common impurities listed above, colchicine may alsocomprise other structurally unidentified impurities. In fact,conventional forms of colchicine may comprise as much as 5% of totalimpurities, determined chromatographically as described below. Such highlevels of impurities in conventional colchicine pose several problems.The impurities may cause side effects in patients taking dosage formscomprising conventional colchicine. For example,N-deacetyl-N-formyl-colchicine (Impurity A, also known as Gloriosine) istumorigenic and has been studied as an anti-cancer agent. Thereforereduction in the level of N-deacetyl-N-formyl colchicine found inconvention colchicine is highly desirable. Additionally, high levels ofimpurities can also pose a regulatory challenge for pharmaceuticalcompanies using conventional colchicine in products. For apharmaceutical product comprising an active agent, the United StatesFood and Drug Administration (FDA) requires “qualification” or toxicityinformation for any impurity that is greater than the InternationalConference on Harmonization (ICH) qualification threshold of 0.15% perindividual impurity in the active agent substance or 1.0% in the dosageform. Thus, there is a regulatory benefit for a pharmaceutical companyto market pharmaceutical products comprising active agents comprisinglow levels of individual impurities or total impurities in the activeagent substance as well as in the dosage form. As a result, for apharmaceutical composition comprising colchicine, it is in the bestinterest of both the pharmaceutical company and the patient thatimpurities be minimized, if possible, in the colchicine and incolchicine compositions or dosage forms.

The ultrapure colchicine disclosed herein comprises no more than (NMT)about 3.0%; specifically NMT about 2.0%, or more specifically, NMT about1.0%, or even more specifically NMT about 0.5% of total impurities. Inone embodiment, “total impurities” includes the common impurities,Impurities A through F, as well as all structurally unidentifiedimpurities eluting within 1.5 times the retention time of colchicineusing an HPLC method as described in more detail below. In anotherembodiment, other HPLC and UPLC methods, for example, as described inmore detail below, can be used to quantify the level of totalimpurities.

The ultrapure colchicine may also comprise low levels of individualimpurities. In one embodiment, the ultrapure colchicine comprises NMTabout 2.0%, specifically NMT about 1.5%; more specifically NMT about1.0%; or yet more specifically, NMT about 0.5%, or even morespecifically, NMT about 0.15%, or still more specifically, NMT about0.10%, of any individual impurity. The impurity can be Impurity A,Impurity B, Impurity C, Impurity D, Impurity E, Impurity F, or anunidentified impurity.

In an embodiment, the ultrapure colchicine comprises no more than (NMT)about 3.0% total impurities and NMT about 0.10% N-deacetyl-N-formylcolchicine.

In another embodiment, the ultrapure colchicine comprises NMT about 3.0%total impurities; NMT about 0.1% per individual impurity of Impurity A,Impurity C, Impurity D, and Impurity E; NMT about 0.15% Impurity F; andNMT about 2.0% of Impurity B.

Not wishing to be bound by theory, it is postulated that theconformational isomer, Impurity B, is in equilibrium with the activecolchicine such that even after purification of the colchicine to about0.5% Impurity B, the purified colchicine re-equilibrates to a level ofImpurity B around about 1% to about 1.3%.

The level of an individual impurity or of total impurities in colchicinemay be determined by any suitable analytical method known in the art. Inone embodiment, the impurity levels are determined using a highperformance liquid chromatography (HPLC) assay, for example, the HPLCmethod described in the Colchicine Official Monograph USP30/NF25, hereinfully incorporated by reference.

Exemplary conditions for HPLC or ultra performance liquid chromatography(UPLC) assays that can be used for the impurity analysis of colchicineor of a colchicine pharmaceutical product are listed in Table 2.

TABLE 2 Exemplary HPLC Conditions For Colchicine Purity AnalysisUSP30/NF25 Colchicine Official Monograph Method HPLC Method UPLC MethodMobile phase 0.5 Molar KH₂PO₄ in pH 7.2 10 mM pH 4.5 AmmoniumMethanol:Water (65:45, Phosphate Acetate v:v), pH adjusted to 5.5Buffer:methanol Buffer:MeOH with H₃PO₄ (MeOH) Gradient Gradient ColumnOctylsilyl silica gel, 4.6 mm × Zorbax SBC(18) 4.6 × Acquity GEH C18 25cm, 5 micron 250 mm 2.1 × 100 mm, 1.7 um Flow rate 1.0 mL/min 1.0 mL/min0.25 mL/min Column Temp Ambient Ambient 30 C. +/− 2 C. Detection 254nanometers (nm) 246 nm 246 nm Injection 20 microliters (μL) 75 μL 7 μLvolume Sample Conc. 0.006 mg/mL 0.120 mg/ml 0.012 mg/ml Run time 15minutes (min) 46 min 25 min

When using one of the above HPLC conditions in Table 2 for colchicinepurity analysis, the relative retention time (RRT) of an impurity can becalculated by the following formula:

RRT of an impurity=RT of the impurity/RT of colchicine,

where RT stands for retention time of the impurity or the colchicine atthe particular conditions used in the assay.

In one embodiment, using the HPLC method in Table 2, the retention time(RT) of colchicine is about 7 minutes and the relative retention times(RRTs) of the common impurities eluting within 1.5 times the retentiontime for colchicine are listed in Table 2A:

TABLE 2A Relative Retention Times (RRTs) of the Common ImpuritiesImpurity ID RRT N-deacetyl-N-formyl colchicine - Impurity A 0.94Conformational isomer - Impurity B 0.8 β-Lumicolchicine - Impurity C 1.2Colchicoside - Impurity D 0.4 3-O-demethyl colchicine - Impurity E 0.7

In one embodiment, the percent of a particular impurity is calculated bydividing the response (peak area) of the impurity peak by the sum of allresponses (total peak area of all peaks, including the colchicine peakand all common and unidentified impurity peaks) eluting within 1.5 timesthe retention time for colchicine in the HPLC assay and multiplying theresult by 100%.

In one embodiment, the level (%) of total impurities is calculated bydividing the sum of responses of any peaks other than that due tocolchicine eluting within 1.5 times the retention time for colchicine bythe sum of all responses eluting in the HPLC assay and multiplying theresult by 100%.

An additional HPLC method for determining the level of impurities otherthan Impurity F in colchicine or in colchicine pharmaceutical productshas been developed and validated for use as an alternative to themethods in Table 2 above. The method is shown in Table 3A below.

TABLE 3A Quantitative HPLC Method for determining all impurities otherthan impurity F in colchicine and colchicine pharmaceutical products.Quantitative HPLC Method for colchicine and colchicine products. Mobilephase pH 4.5 Ammonium Acetate Buffer: methanol Gradient Column WatersXBridge C18, 250 mm × 4.6 mm, 5 μm particle size Flow rate 0.9 mL/minColumn Temp 10 ± 3.5 C. (for column)/10 ± 2 C. (for sample) Detection246 nm Injection volume 75 μL Sample Conc. 0.16 mg/ml Run time 60 min

In the quantitative HPLC method of Table 3A, the retention time (RT) ofcolchicine is about 24 minutes and the relative retention times (RRTs)of the common impurities for colchicine are listed in Table 3B:

TABLE 3B Relative Retention Times (RRTs) of the Common ImpuritiesImpurity ID RRT N-deacetyl-N-formyl colchicine - Impurity A 0.93Conformational isomer - Impurity B 0.82 β-Lumicolchicine - Impurity C1.76 Colchicoside - Impurity D 0.18 3-O-demethyl colchicine - Impurity E0.52 2-O-demethyl colchicine 0.54 Gamma-Lumicolchicine 1.37

The percentage of individual impurities in the sample solution iscalculated as follows:

${\% \mspace{14mu} {Impurity}} = {\frac{r_{1}}{r_{S}} \times \frac{W_{S}({mg})}{100\mspace{14mu} {mL}} \times \frac{6.0\mspace{14mu} {mL}}{100\mspace{14mu} {mL}} \times \frac{2.0\mspace{14mu} {mL}}{100\mspace{14mu} {mL}} \times P \times \left( \frac{100 - {\% \mspace{14mu} {RS}_{S}} - {\% \mspace{14mu} W_{S}}}{100} \right) \times \frac{200\mspace{14mu} {mL}}{{SW}\; ({mg}) \times \left( \frac{100 - {\% \mspace{14mu} {RS}_{u}} - {\% \mspace{14mu} W_{u}}}{100} \right)} \times \frac{100\%}{RRF}}$

Where:

r_(s)=The area response of the Colchicine peak in the Working StandardSolution.

r_(i)=The area response of the impurity peak in the Sample Solution

P=% Purity of the Colchicine Reference Standard divided by 100%.

SW=Weight of Sample taken for Sample Preparation

W_(s)=Weight of Colchicine in the Stock Standard Solution

RRF=Relative Response Factor for specified and unspecified impurities,1.0

% RS_(s/u)=Percent of Residual Solvents in the ColchicineStandard/Sample

% W_(s/u)=% Water in the Colchicine Standard/Sample

To date, the impurity colchiceine (Impurity F or 10-O-DemethylColchicine “10-DMC”) has been typically analyzed by a qualitativecolorimetric test described in the Colchicine Official MonographUSP30/NF25 using ferric chloride solution, rather thanchromatographically. The standard for acceptable levels of Impurity Fhas been absence of production of a definite green color in a solutionof colchicine.

However, a chromatographic method has been developed for thedetermination of Impurity F (Colchiceine or 10-O-Demethyl Colchicine“10-DMC”). The chromatographic conditions are as follows:

TABLE 3C HPLC parameters for Colchiceine determination HPLC System: HPLCequipped with a pump, auto sampler, variable wavelength detector and asuitable data acquisition system. Column: Phenomenex Gemini C18 150 mm ×4.6 mm 5 μm, 110 Å Detection: 245 nm Flow Rate: About 1.5 mL/minInjection Volume: 50 μL Temperature: Column: 10° C. ± 3.5° C. Sample: 5°C. ± 2° C. Needle Rinse Setting: Double Needle Wash: Water:Acetonitrile(50:50) Digital Filter Response: 1.0 Sampling Rate: 5.0 Resolution: 1.2Mobile Phase: pH 4.5 Buffer Solution:Acetonitrile (75:25) Run Time:About 7 minutes for Standard About 20 minutes for first Blank andSamples

The LQL level for 10-DMC in this method is 0.776304 μg/mL. The amount of10-DMC expressed in percent of Colchicine is calculated as follows:

${\% \mspace{14mu} {Purity}} = {\frac{r_{i}}{r_{s}} \times \frac{{W_{s}({mg})} \times P}{400\mspace{14mu} {mL}} \times \left( \frac{100 - {\% \mspace{14mu} {RS}_{s}} - {\% \mspace{14mu} W_{s}}}{100} \right) \times \frac{3.0\mspace{14mu} {mL}}{100\mspace{14mu} {mL}} \times \frac{50\mspace{14mu} {mL}}{{W_{u}({mg})} \times \left( \frac{100 - {\% \mspace{14mu} {RS}_{u}} - {\% \mspace{14mu} W_{u}}}{100} \right)} \times \frac{100\%}{RRF}}$

Where:

r_(i)=The peak area response of 10-DMC in the Sample Solution

r_(s)=The peak area response of Colchicine in the Working StandardSolution

W_(s)=The weight of Colchicine in the Stock Standard Preparation

W_(u)=The weight of Colchicine in the Sample Preparation

P=Standard purity factor expressed as labeled (% Purity/100)

% RS_(s/u)=Percent of Residual Solvents in the ColchicineStandard/Sample

% W_(s/u)=% Water in the Colchicine Standard/Sample

RRF=Relative response factor for 10-DMC=0.88

Ultrapure colchicine may be obtained by various purification methodsstarting from colchicine-containing botanical extracts, conventionalcolchicine, or other partially-purified forms of colchicine. In someembodiments, the colchicine is purified from a botanical source. Thebotanical source can be any capable of providing colchicine,conventional or ultrapure, in quantities suitable for commercialpharmaceutical product manufacture

The literature from 1884-1997 on methods of isolation and purificationof colchicine from various botanic sources, including for example C.autumnale corms or leaves and species of Gloriosa has been reviewed.(Kiselev & Yavich, 1991, “METHODS OF ISOLATING ALKALOIDS OF THECOLCHICINE SERIES”; Plenum Publishing Co., English translation ofarticle from Khimiya Prirodnykh Soedinenii, No. 5, pp. 592-600,September-October, 1990.). Kiseleve & Yavich also review reports in theliterature of impurities detected in colchicine stored for varioustimes, as follows. In an article published in 1944 it was reported thatchromatography of colchicine corresponding to the requirements of theUSP of that time showed the presence of 5% of impurities and variousamount of individual impurities. An article published in 1952 reportedthat colchicine meeting the requirements of the USP contained about 4%of 3-demethylcolchicine. A 1953 article reported 1.5% ofN-formyldeacetylcolchicine isolated and the presence of otheraccompanying alkaloids in a pharmacopeial sample of colchicine. Aninvestigation of a commercial sample by high-resolution liquidchromatography, published in 1986, reported finding 93.4% of colchicine,2.9% of N-formyldeactylcholchicine, 1.8% of 17-hydroxycolchicine, and0.84% of an unknown substance.

Walaszik et al. describes a process of incorporating carbon 14 into C.autumnale plants and isolating radioactive colchicine from theradioactive plants (See Walaszik et al., Science (1952) 116:225-227).However, the level of impurities in the isolated radioactive colchicineis not disclosed.

The purification methods disclosed herein produce ultrapure colchicinecomprising very low levels of total impurities or individual impurities.In one embodiment, ultrapure colchicine may be obtained fromconventional colchicine obtained commercially or produced using solventextraction of appropriate botanic material as described in more detailbelow.

In one embodiment, conventional colchicine may be obtained by isolatingcolchicine from a colchicine chloroform extract. The extract is washedwith a mixture of purified water, sodium hydroxide solution, sodiumchloride solution and acetic acid. The washed extract is filtered andthe resulting concentrate is distilled in two steps, first usingmethanol, and second using ethyl acetate. The resulting distillate iscrystallized. Ethyl acetate can be used to isolate and wash thecrystallized colchicine, which is then dried to yield conventionalcolchicine. The conventional colchicine comprises more than about 3.0%but no more than 5% total impurities.

The conventional colchicine may be used directly in a colchicinecomposition comprising a pharmaceutically acceptable excipient. It mayalso be used for further purification to obtain ultrapure forms ofcolchicine.

In one embodiment of a method of making ultrapure colchicine, theconventional colchicine may be subjected to column chromatography toobtain a purified colchicine concentrate, distilling the purifiedcolchicine concentrate to obtain a colchicine distillate, andcrystallizing ultrapure colchicine from the colchicine distillate. Themethod can further comprise washing the crystallized ultrapurecolchicine with a solvent and drying. The ultrapure colchicine comprisesno more than about 3.0% of total impurities.

In one embodiment, the column chromatography is carried out usingmethylene chloride as solvent on a column of neutral alumina. Othersolvents or chromatographic media may be used, provided that impuritiesare removed from the conventional colchicine to the desired level. Inanother embodiment, distillation of the purified colchicine concentrateis carried out using ethyl acetate. Other organic solvents can be used,provided that impurities are removed from the purified colchicineconcentrate. In yet another embodiment, the solvent used to wash thecrystallized ultrapure colchicine is ethyl acetate.

In another embodiment, a method of making ultrapure colchicine comprisessubjecting colchicine comprising more than about 3.0% total impuritiesto neutral alumina column chromatography to obtain a colchicineconcentrate; distilling the colchicine concentrate with ethyl acetate toobtain a colchicine distillate; crystallizing a purified colchicine fromthe colchicine distillate in ethyl acetate; washing the purifiedcolchicine with ethyl acetate to obtain a washed purified colchicine;and drying the washed purified colchicine to obtain ultrapurecolchicine, wherein the ultrapure colchicine comprises no more thanabout 3.0% total impurities.

In one embodiment, the ultrapure colchicine obtained in any of the abovemethods comprises no more than about 2.0% total impurities. In anotherembodiment, the ultrapure colchicine comprises no more than about 1.5%total impurities. In another embodiment, the ultrapure colchicinecomprises no more than about 1.0% total impurities. In anotherembodiment, the ultrapure colchicine comprises no more than about 0.5%total impurities. In yet another embodiment, the ultrapure colchicinecomprises no more than about 0.5% per individual impurity of Impurity A,Impurity B, Impurity C, Impurity D, Impurity E, or Impurity F. In stillanother embodiment, the ultrapure colchicine comprises no more thanabout 0.15% per individual impurity of Impurity A, Impurity C, ImpurityD, Impurity E, or Impurity F. In another embodiment, the ultrapurecolchicine comprises no more than about 1.0% of total unidentifiedimpurities. In yet another embodiment, the ultrapure colchicinecomprises no more than about 0.5% of total unidentified impurities. Inyet another embodiment, the ultrapure colchicine comprises no more than1.0% of Impurity B, and no more than 0.1% per individual impurity of anyof Impurity A, Impurity C, Impurity D, Impurity E, and Impurity F.

The above methods of making ultrapure colchicine are only examples ofsuitable methods for its preparation.

The colchicine compositions disclosed herein comprise colchicine and apharmaceutically acceptable excipient. In one embodiment, the colchicinecomposition comprises 0.1 wt % to 10 wt % colchicine, specifically 0.1wt % to 5 wt % colchicine. In one embodiment, the colchicine in thecolchicine compositions is ultrapure colchicine. The compositionscomprising ultrapure colchicine are stable and provide enhanced safetyto patients taking the compositions because the patients are ingestingfewer impurities. In particular, the colchicine compositions containsubstantially lower levels of the tumorigenic compoundN-deacetyl-N-formyl colchicine.

The pharmaceutically acceptable excipient in the colchicine compositionmay be a filler (or diluent), a binder, a disintegrant, a lubricant, ora combination comprising two or more of the foregoing excipients.

In one embodiment, the pharmaceutically acceptable excipient comprises afiller. Exemplary fillers may be one or more compounds which are capableof providing compactability and good flow. Exemplary fillers includemicrocrystalline cellulose, starch, lactose, sucrose, glucose, mannitol,maltodextrin, sorbitol, dextrose, silicic acid, dibasic calciumphosphate, or a combination comprising at least one of the foregoingfillers. Exemplary lactose forms include lactose monohydrate, NF (FastFlo), lactose spray-dried monohydrate, and lactose anhydrous. Exemplarymicrocrystalline cellulose (MCC) include, for example, AVICEL® PH101 andAVICEL® PH102, which are commercially available from FMC Biopolymer,Philadelphia, Pa. Exemplary dibasic calcium phosphates includedihydrated and anhydrous dibasic calcium phosphates. In one embodiment,the filler is a combination of microcrystalline cellulose and lactosemonohydrate, NF (fast flo).

When present, the amount of the filler in the composition may be about10 wt % to about 99 wt %, or more specifically, about 30 wt % to about90 wt %, or even more specifically, about 50 wt % to about 90 wt %, orstill more specifically, about 70 wt % to about 85 wt %, based on thetotal weight of the composition. In one embodiment, the total amount ofthe filler is about 82 wt %, based on the total weight of thecomposition

In one embodiment, the pharmaceutically acceptable excipient comprises abinder. Binders may be used to impart cohesive qualities to aformulation, for example, a tablet formulation, and thus ensure that thetablet remains intact after compaction. Exemplary binders includestarches (for example, Starch 1500® or pregelatinized starch),alignates, gelatin, carboxymethylcellulose, sugars (for example,sucrose, glucose, dextrose, and maltodextrin), polyethylene glycol,waxes, natural and synthetic gums, polyvinylpyrrolidone, and cellulosicpolymers (for example, microcrystalline cellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose, andhydroxyethyl cellulose) and combinations comprising one or more of theforegoing binders. In one embodiment, the binder is starch, or morespecifically, pregelatinized starch.

When present, the amount of the binder may be about 10 wt % to about 99wt %, or more specifically, about 10 wt % to about 50 wt %, or even morespecifically, about 10 wt % to about 20 wt %, based on the total weightof the composition. In one embodiment, the amount of the binder is about14 wt %, based on the total weight of the composition.

In another embodiment, the pharmaceutically acceptable excipientcomprises a disintegrant. Disintegrants are used to facilitatedisintegration or “breakup” of a composition, for example, a tablet,after administration. Exemplary disintegrants include sodium starchglycolate, sodium crosscarmelose (cross-linked carboxy methylcellulose), crosslinked polyvinylpyrrolidone (PVP-XL), anhydrous calciumhydrogen phosphate, agar-agar, potato or tapioca starch, alginic acid,or a combination comprising one or more of the foregoing disintegrants.

When present, the disintegrant may be present in an amount of about 0.1to 30 wt %, or more specifically, about 1 to 20 wt %, or even morespecifically, about 1 to 10 wt %, based on the total weight of thecomposition. In one embodiment, the amount of the disintegrant is about4.5 wt %, based on the total weight of the composition.

In another embodiment, the pharmaceutically acceptable excipientcomprises a lubricant. Generally, a lubricant is added just beforetableting, and is mixed with the rest of the composition for a minimumperiod of time to obtain good dispersal. Exemplary lubricants includemagnesium stearate, calcium stearate, zinc stearate, stearic acid, talc,glyceryl behenate, polyethylene glycol, polyethylene glycol,polyethylene oxide, sodium lauryl sulfate, magnesium lauryl sulfate,sodium oleate, sodium stearyl fumarate, DL-leucine, colloidal silica, ora combination comprising one or more of the foregoing lubricants. In oneembodiment, the lubricant is magnesium stearate, calcium stearate, orzinc stearate.

When present, the lubricant may be present in an amount of about 0.01 wt% to about 10 wt %, or more specifically, about 0.1 wt % to about 5 wt%, or even more specifically, about 0.1 wt % to about 1 wt %, based onthe total weight of the composition. In one embodiment, the amount ofthe lubricant is about 0.6 wt %, based on the total weight of thecomposition.

If desired, the composition may optionally comprise small amounts ofnontoxic auxiliary substances such as wetting or emulsifying agents, orpH buffering agents, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, sodium laurylsulfate, dioctyl sodium sulfosuccinate, and polyoxyethylene sorbitanfatty acid esters.

In one embodiment, a composition comprises an ultrapure colchicine,wherein the ultrapure colchicine comprises no more than 3.0% of totalimpurities, a filler, a binder, and a disintegrant.

In another embodiment, a colchicine composition comprises about 0.6 mgAcolchicine; about 14 mg pregelatinized starch; about 22 mgmicrocrystalline cellulose; about 4.3 mg sodium starch glycolate; about0.6 mg magnesium stearate; and an amount of lactose monohydrate suchthat the colchicine composition has a total weight of about 100 mg. Inone embodiment, the colchicine in the colchicine composition isultrapure colchicine.

In an embodiment, a colchicine composition comprises colchicine; and apharmaceutically acceptable excipient; wherein the colchicinecomposition comprises no more than about 3.5% total impurities,specifically no more than about 3.0% total impurities, more specificallyno more than about 2.0% total impurities, or yet more specifically nomore than about 1.0% total impurities. In some of these embodiments,specific limitations on the levels of individual impurities are also metby the colchicine composition. In addition to containing no more than aparticular maximum level of total impurities, the colchicine compositioncan comprise not more than about 0.42% Impurity A, Impurity C, ImpurityD, Impurity E, or Impurity F, and not more than about 2.0% Impurity B;specifically not more than about 0.2% Impurity A, Impurity C, ImpurityD, Impurity E, or Impurity F, and not more than about 1.5% Impurity B;and more specifically not more than about 0.15% Impurity A, Impurity C,Impurity D, Impurity E, or Impurity F, and not more than about 1.1%Impurity B. In one embodiment the colchicine composition comprises nomore than 3.5% total impurities, no more than 0.42% Impurity A, and nomore than 2.0% Impurity B. The pharmaceutically acceptable excipient canbe one or more discussed previously herein.

In one embodiment, a colchicine composition comprises colchicine and apharmaceutically acceptable excipient; wherein the colchicinecomposition comprises no more than about 3.5% total impurities. In someembodiments, the colchicine composition comprises ultrapure colchicineand total impurities in the ultrapure colchicine comprise no more thanabout 3.0%, or specifically no more than about 2.0%, or morespecifically no more than about 1.5%, or yet more specifically, no morethan about 1.0%. In addition to containing no more than a particularmaximum level of total impurities, the ultrapure colchicine in thecolchicine composition can comprise not more than about 0.15% ImpurityA, Impurity C, Impurity D, Impurity E, or Impurity F, and not more thanabout 2.0% Impurity B; specifically not more than about 0.10% ImpurityA, Impurity C, Impurity D, or Impurity E; not more than about 0.15%Impurity F, and not more than about 1.5% Impurity B. In one embodimentthe colchicine composition comprises ultrapure colchicine comprising nomore than 0.10% Impurity A, no more than about 0.15% Impurity F, and nomore than 2.0% Impurity B. The pharmaceutically acceptable excipient canbe one or more discussed previously herein.

In one embodiment, a colchicine composition comprises colchicine; afiller; a binder; and a disintegrant; wherein the colchicine compositioncomprises no more than about 3.5% total impurities. In some embodiments,the colchicine composition comprises ultrapure colchicine and totalimpurities in the composition comprise no more than about 3.5%, orspecifically no more than about 3.0%, more specifically no more thanabout 2.0%, or yet more specifically, no more than about 1.0%; withindividual impurity levels of not more than about 0.42% for Impurity A,Impurity C, Impurity D, Impurity E, or Impurity F, and not more thanabout 2.0% for Impurity B. or specifically with individual impuritylevels of not more than about 0.10% for Impurity A, Impurity C, ImpurityD, Impurity E, or Impurity F, and not more than about 2.0% for ImpurityB.

In one embodiment, the percent total impurities in the composition isdetermined in an HPLC assay as described in Colchicine OfficialMonograph USP30/NF25 as 100% times a sum of responses of any peaks otherthan that due to colchicine, eluting within 1.5 times the retention timefor colchicine, relative to a sum of responses of all peaks elutingwithin 1.5 times the retention time for colchicine and the percent of anindividual impurity in the composition is determined in an HPLC assay asdescribed in Colchicine Official Monograph USP30/NF25 as 100% times theresponses of the impurity peak relative to a sum of responses of allpeaks eluting within 1.5 times the retention time for colchicine.

In yet another embodiment, the percent total and/or individualimpurities in the composition is determined in an HPLC assay or UPLCassay in accordance with the methods described in Table 2 or Table 3A or3C.

In one embodiment, any of the colchicine compositions described above isin the form of a tablet. As used herein, the term “tablet” means acompressed pharmaceutical dosage form of any shape or size. The tabletsdescribed herein may be obtained from the compositions comprisingcolchicine and a pharmaceutically acceptable excipient. Any of thecolchicine compositions can be in the form of any other dosage formknown in the art, specifically, any oral dosage form, for example acapsule.

Either wet or dry granulation of a colchicine composition may be usedprior to compressing the composition into tablets, or direct compressioncan be used.

In one embodiment, wet granulation is used to prepare wet granulescomprising colchicine. A granulating liquid is used in wet granulationprocess. Both aqueous and non-aqueous liquids may be used as thegranulating liquid. In one embodiment, the granulating liquid is anaqueous liquid, or more specifically, de-ionized water. In anembodiment, the colchicine is ultrapure colchicine.

The amount of the granulating liquid used may depend on many factors,for example, the type of the granulating liquid, the amount of thegranulating liquid used, whether a hygroscopic excipient is used, thenature of the active agent, and the active agent loading. In oneembodiment, the amount of the granulating liquid is in the range ofabout 5 wt % to about 50 wt %, or more specifically, about 10 wt % toabout 40 wt %, based on the dry weight of the granulating particlesprior to wet granulation.

Wet granulation time is generally about 5 to 60 minutes. In oneembodiment, the colchicine particles and suitable excipients are mixedwith the granulating liquid for a period of about 5 to about 45 minutes,or more specifically, about 5 to about 35 minutes. For a small scale,the mixing time is about 1 to about 20 minutes, or more specifically, 3to 10 minutes. Wet granulation is generally performed at temperaturesbetween about 20° C. to about 35° C., or more specifically, at roomtemperature (about 25° C.).

Any equipment may be used to contact the granulating liquid with thecolchicine and the excipients as long as uniform distribution of thegranulating liquid is achieved. For example, small-scale production canbe achieved by mixing and wetting the ultrapure colchicine and theexcipients in mortars or stainless steel bowls, while for largerquantities, V-blenders with intensifier bars, planetary mixers, rotarygranulators, high shear granulators, and fluid-bed granulation equipmentmay be used. In one embodiment, the granulator is a high sheargranulator.

In one embodiment, a method of making a colchicine composition compriseswet granulating colchicine with a pharmaceutically acceptable excipientto obtain wet granules, and mixing the granules with a second excipientto obtain a colchicine composition. In one embodiment, thepharmaceutically acceptable excipient comprises a mixture of a fillerand a binder. In another embodiment, the mixture of the filler and thebinder comprises pregelatinized starch, lactose monohydrate, andmicrocrystalline cellulose. In another embodiment, de-ionized water isused as the granulating liquid. In some embodiments, the colchicine isultrapure colchicine. In an embodiment, the second excipient mixed withthe granules is a disintegrant. The colchicine compositions can containabout 0.1 wt % to about 10 wt %, or more specifically, about 0.1 wt % toabout 1 wt %, of colchicine, based on the total weight of the colchicinecomposition.

In an embodiment, the method of making a composition comprises wetgranulating colchicine with a pharmaceutically acceptable excipient toobtain wet granules, and mixing the granules with a disintegrant toobtain a colchicine composition. In some embodiments, the method furthercomprises drying the mixture. In another embodiment, the wet granulesare dried to obtain dried granules; and then the dried granules aremixed with a disintegrant to obtain the composition. In anotherembodiment, the dried granules can be milled to obtain milled granulesbefore mixing the milled dried granules with the disintegrant. In oneembodiment, greater than 50% of the milled granules pass through a 45micron sieve or mesh screen. The method can further comprise mixing thecolchicine composition with a lubricant to obtain a tableting blend orcompressing the tableting blend to obtain a tablet. The method canfurther comprise coating the tablet.

In one embodiment, a method of making a colchicine composition compriseswet granulating ultrapure colchicine with a pharmaceutically acceptableexcipient to obtain wet granules; drying the wet granules to obtaindried granules; milling the dried granules to obtain milled granules;and mixing the milled granules with a disintegrant to obtain thecomposition. The ultrapure colchicine can comprise no more than about3.0% total impurities, with no more than about 0.10% Impurity A, no morethan about 0.15% Impurity F, and no more than about 2.0% Impurity B.

In another embodiment, a method of making a colchicine tablet compriseswet granulating colchicine with a pharmaceutically acceptable excipientto obtain wet granules; drying the wet granules to obtain driedgranules; milling the dried granules to obtain milled granules; mixingthe milled granules with a disintegrant to obtain the composition;mixing the composition with a lubricant to obtain a tableting blend; andcompressing the tableting blend to obtain a colchicine tablet.

In some embodiments, the wet granules are dried to obtain dried granulesbefore mixing with a second excipient, for example a disintegrant. Wetgranules can be dried by any suitable means to remove the granulatingliquid and to form dried granules containing colchicine and thepharmaceutically acceptable excipient. The conditions and duration ofdrying depend on factors such as the liquid used and the weight of thegranulating particles. Examples of suitable drying methods include, butare not limited to, tray drying, forced air drying, microwave drying,vacuum drying and fluid bed drying.

The extent of drying may be determined by visual observation and manualmanipulation, as is common in the art. The extent of drying may also bedetermined by sieve analysis, moisture measurements, such as loss ondrying (LOD) or other suitable methods. In one embodiment, wet granulesare dried until the granules lose less than 5 weight percent (wt %), ormore specifically, 3 wt % upon drying at 105° C. based on the totalweight of the dried granules prior to drying (or LOD of less than 3 wt%).

After drying, dried granules may be mixed directly with an excipient,for example, a filler, a binder, a disintegrant, or a lubricant, forfurther processing. Alternatively, dried granules may optionally besubjected to additional processing steps prior to mixing with theexcipient. For example, dried granules may be sized to reduce particlesize prior to mixing with an excipient. Exemplary sizing operationsinclude milling or sieving. Any suitable equipment for reducing theparticle size may be used in the present invention. In one embodiment,the dried granules are milled to obtain milled granules so that at least50% of the milled granules pass through a 45 micron mesh screen.

Suitable excipients may be added extragranularly and mixed with thegranules to form colchicine compositions. As used herein, the term“extragranular” or “extragranularly” means that the referenced material,for example, a suitable excipient, is added or has been added as a drycomponent after wet granulation. In one embodiment, a disintegrant and alubricant, in that sequence, are added extragranularly to the granulesand mixed to form a blend. The blend may be encapsulated directly intocapsule shells, for example, hard gelatin shells, to form capsuleformulations. Alternatively, the blend may be compressed into tablets.In some embodiments, the granules are dried granules or milled, driedgranules. In some embodiments, the colchicine is ultrapure colchicine.

Mixing can be carried out for a sufficient time to produce homogeneousmixtures or blends. Mixing may be accomplished by blending, stiffing,shaking, tumbling, rolling, or by any other method to achieve ahomogeneous blend. In some embodiments, the components to be mixed arecombined under low shear conditions in a suitable apparatus, such as aV-blender, tote blender, double cone blender or any other apparatuscapable of functioning under low shear conditions.

The homogenous mixtures or blends are then compressed using any methodsuitable in the industry.

The colchicine tablets prepared from the above described methods exhibitacceptable physical characteristics including good friability andhardness. The colchicine tablets disclosed herein have friability in therange of about 0% to 3%, specifically about 0 to 1%, more specifically0% to 0.5%

The colchicine tablet can be coated. Coating the tablet may be performedby any known process. A coating for the colchicine tablet disclosedherein can be any suitable coating, such as, for example, a functionalor a non-functional coating, or multiple functional or non-functionalcoatings. By “functional coating” is meant to include a coating thatmodifies the release properties of the total formulation, for example, asustained-release coating. By “non-functional coating” is meant toinclude a coating that is not a functional coating, for example, acosmetic coating. A non-functional coating can have some impact on therelease of the active agent due to the initial dissolution, hydration,perforation of the coating, etc., but would not be considered to be asignificant deviation from the non-coated composition.

In one embodiment, the tablet is coated with a non-functional coating.The coating can be a white or colored OPADRY® or OPADRY® II (bothavailable from Colorcon, West Point, Pa.), optionally with additionalingredients such as carnauba wax, plasticizers, opacifiers, colorants,and antioxidants. In one embodiment, the coating comprises OPADRY® IIand carnauba wax.

In an embodiment, a colchicine composition comprises about 0.6 mgAcolchicine; about 12 to about 16 mg pregelatinized starch; about 20 toabout 24 mg microcrystalline cellulose; about 3.9 to about 4.7 mg sodiumstarch glycolate; about 0.5 to about 0.7 mg magnesium stearate; and anamount of lactose monohydrate such that the colchicine dosage form has atotal weight of about 100 mg. In some embodiments the colchicinecomposition comprises about 0.6 mgA colchicine, about 14 mgpregelatinized starch, about 22 mg microcrystalline cellulose, about 4.3mg sodium starch glycolate, about 0.6 mg magnesium stearate, and anamount of lactose monohydrate such that the colchicine dosage form has atotal weight of about 100 mg. The colchicine composition can be in theform of a tablet. In some embodiments the tableted composition furthercomprises a coating comprising Opadry® II and carnauba wax. In anembodiment, the colchicine is ultrapure colchicine.

In one embodiment, a colchicine composition comprising ultrapurecolchicine is formulated into an immediate-release formulation. By“immediate-release” is meant a conventional or non-modified release inwhich greater than or equal to about 75% of the active agent is releasedwithin two hours of administration, specifically within one hour ofadministration.

Active agent release from a pharmaceutical formulation can be analyzedin various ways. One exemplary test is in vitro dissolution. Adissolution profile is a plot of the cumulative amount of active agentreleased from a formulation as a function of time. A dissolution profilecan be measured utilizing the Drug Release Test <724>, whichincorporates standard test USP 26 (Test <711>). A profile ischaracterized by the test conditions selected such as, for example,apparatus type, shaft speed, temperature, volume, and pH of thedissolution medium. More than one dissolution profile may be measured.For example, a first dissolution profile can be measured at a pH levelapproximating that of the stomach, and a second dissolution profile canbe measured at a pH level approximating that of one point in theintestine or several pH levels approximating multiple points in theintestine.

In one embodiment, the immediate-release colchicine composition exhibitsa dissolution profile such that at ten minutes after combining thecomposition with 500 ml of purified water at 37° C.±0.5° C. according toUSP 28 <711> Apparatus 1 (basket), 100 rpm speed, about 90 to about 100wt. % of the total amount of active agent is released; specifically at30 minutes after combining the composition with the dissolution medium,about 95 to about 100 wt. % of the total amount of colchicine isreleased; and more specifically at one hour after combining thecomposition with the dissolution medium, about 98 to about 100 wt. % ofthe total amount of colchicine is released.

Potency, or the amount of active colchicine present in a batch ofcolchicine, can be determined as described in Colchicine OfficialMonograph USP 30/NF 25 by comparing an assay sample to a colchicinereference standard (e.g., USP Colchicine RS) sample in thechromatographic assay described in Colchicine Official MonographUSP30/NF25 The quantity of active colchicine in the assay sample, in mg,of C₂₂H₂₅NO₆ is calculated by the formula: 10C(r_(U)/r_(S)), in which Cis the concentration, in μg per mL, of the colchicine reference standardsample; and r_(U) and r_(S) are the colchicine peak responses obtainedfrom the assay sample and the colchicine reference standard sample,respectively.

In another embodiment, potency of a batch of colchicine can bedetermined using the HPLC Potency assay described in the table below bycomparing an assay sample to a colchicine reference standard.

HPLC Potency Assay B Mobile phase 50 mM Potassium PhosphateBuffer:methanl (45:55), pH 5.5 ± 0.05 Column Phenomenex Luna C8(2), 4.6mm × 25 cm, 5 μm Flow rate 1.0 mL/min Column Temperature AmbientDetection 254 nm Injection volume 20 uL Sample Conc. 0.120 mg/ml Runtime 15 min

The quantity, in percentage, of C₂₂H₂₅NO₆ (active colchicine), on ananhydrous, solvent free basis, in the colchicine assay sample iscalculated by the formula:

${\% \mspace{14mu} {Purity}} = {\frac{r_{u}}{r_{s}} \times \frac{{W_{s}({mg})} \times P \times \left( \frac{100 - M_{s} - S_{s}}{100} \right)}{500\mspace{14mu} {ml}} \times \frac{{PV}({ml})}{{VF}({ml})} \times \frac{{VF}_{1}({ml})}{{{SW}({mg})} \times \left( \frac{100 - M_{u} - S_{u}}{100} \right)} \times \frac{{VF}_{2}({ml})}{{PV}_{1}({ml})} \times 100}$

Where:

r_(u)=The peak area of colchicine in the working sample solutionr_(s)=The peak area of colchicine in the working standard solutionW_(s)=The weight of colchicine in the standard preparationP=Standard purity factor expressed as labeled % PurityM_(s)=Moisture factor in standard expressed as % MoistureS_(s)=Solvent factor in standard expressed as % SolventPV=Pipet volume used for the working standard solutionVF=Volumetric flask used for the working standard solutionSW=Sample weight in the stock sample solutionVF₁=Volumentric flask used for the stock sample solutionM_(u)=Moisture factor in sample expressed as % MoistureS_(u)=Solvent factor in sample expressed as % SolventVF₂=Volumentric flask used for the working sample solutionPV₁=Pipet volume used for the working sample solution.

Alternatively, potency of a batch of colchicine can be determined bycomparing an assay sample to a colchicine reference standard in yetanother HPLC assay as follows:

HPLC Potency Assay C HPLC System: HPLC equipped with a pump,autosampler, variable wavelength detector and a suitable dataacquisition system Column Information: Phenomenex Gemini C18 150 × 4.6mm 5 μm 110 Å Detection: 245 nm Flow Rate: 1.5 mL/minute InjectionVolume: 20 μL Column Temperature: 30° C. ± 3° C. Needle Rinse Setting:Double Sampling Rate: 2.0 Resolution: 1.2 Filter Response: 1.0 DigitalFilter: Enabled Needle Wash/Seal Wash: Methanol:Water (50:50) Run Time:About 15 minutes Mobile Phase: pH 6.0 Buffer Solution (50 mM ofPotassium phosphate and 4 mM of EDTA):Methanol (60:40) Diluent:Water:Methanol (75:25)

The percent purity of Colchicine (C₂₂H₂₅N₀₆), on an anhydrous,solvent-free basis, is calculated as follows:

${\% \mspace{14mu} {Assay}} = {\frac{r_{u}}{r_{s}} \times \frac{{W_{s}({mg})} \times P}{50\mspace{14mu} {mL}} \times \left( \frac{100 - {\% \mspace{14mu} {RS}_{s}} - {\% \mspace{14mu} W_{s}}}{100} \right) \times \frac{5.0\mspace{14mu} {mL}}{100\mspace{14mu} {mL}} \times \frac{500\mspace{14mu} {mL}}{{W_{u}({mg})} \times \left( \frac{100 - {\% \mspace{14mu} {RS}_{u}} - {\% \mspace{14mu} W_{u}}}{100} \right)} \times 100\%}$

Where:

r_(U)=The peak area response of Colchicine in the Sample Solution.

r_(s)=The peak area response of Colchicine in the Working StandardSolution.

W_(s)=The weight of Colchicine in the Stock Standard Preparation.

W_(u)=The weight of Colchicine in the Sample Preparation.

P=Standard purity factor expressed as labeled (% Purity/100).

% RS_(s/u)=Percent of Residual Solvents in the ColchicineStandard/Sample.

% W_(s/u)=% Water in the Colchicine Standard/Sample.

Disclosed herein are also methods of treatment and dosing regimens.

The compositions comprising ultrapure colchicine disclosed herein may beused to treat or prevent a patient's condition such as acute goutyarthritis, chronic gouty arthritis, acute pericarditis, asthma, Behçet'sdisease, cancer, chronic gout (prophylaxis), pseudogout cystic diseasecomprising polycystic kidney disease or cystic fibrosis, demyelinatingdisease of central or peripheral origin, Dupuytren's contracture,Familial Mediterranean fever, glaucoma, idiopathic pulmonary fibrosis,idiopathic thrombocytopenic purpura, inflammatory disorder comprisingrheumatoid arthritis, lentiviral infection, multiple sclerosis,postpericardiotomy syndrome, primary amyloidosis, primary biliarycirrhosis, proliferative vitreoretinopathy, pyoderma gangrenosum,recurrent pericarditis, or a condition in need of enhanced boneformation or bone mineral density.

The traditional dose of colchicine used to treat or prevent an attack ofacute gouty arthritis has been about 1.0 to about 1.2 mgA of colchicine,for example, two tablets each comprising about 0.6 mgA colchicine. Thisdose may be followed by one unit of the composition every hour, or twounits every two hours, until pain is relieved or until diarrhea ensues(“diarrheal dose”). After the initial dose, it is sometimes sufficientto take about 0.6 mgA colchicine every two or three hours. The dosingshould be stopped if there is gastrointestinal discomfort or diarrhea.(Opiates may be needed to control diarrhea.) In subsequent attacks, thepatient should be able to judge his medication requirement accuratelyenough to stop short of his diarrheal dose. The total amount ofcolchicine needed to control pain and inflammation during an attack hasbeen believed to be in the range from about 4 mgA to about 8 mgA. Aninterval of three days between colchicine courses is advised in order tominimize the possibility of cumulative toxicity.

In one embodiment, a method of treating acute gouty arthritis comprisesadministering two colchicine dosage forms each comprising about 0.6 mgAcolchicine at the onset of the acute gout attack, followed by one dosageform every hour for m hours, wherein the value of m is 1 to 8. In oneembodiment, the value of m is 1 to 6. In another embodiment, the valueof m is 1 (total of 3 tablets). In yet another embodiment, the value ofm is 6 (total of 8 tablets). The colchicine in the dosage form can beultrapure colchicine. The dosage form can be any oral dosage form,specifically a tablet.

In another embodiment, a method of treating Familial Mediterranean Fevercomprises administering 1/2 dosage form to four dosage forms daily, eachdosage form comprising about 0.6 mgA colchicine (total of about 0.3 toabout 2.4 mgA colchicine daily). In another embodiment, a method ofprophylactically treating chronic gout comprises administering one-halfdosage form, one dosage form, two dosage forms, or three dosage forms,each dosage form comprising about 0.6 mgA of colchicine, daily. Inanother embodiment, a method of treating Behçet's disease comprisesadministering one dosage form comprising about 0.6 mgA of colchicinetwice daily (total of 2 dosage forms). The colchicine in the dosage formcan be ultrapure colchicine. The dosage form can be any oral dosageform, specifically a tablet.

In one embodiment, a method of treating patients with some but not allof the symptoms of acute gout, chronic gout (prophylaxis), orpseudogout, where the patients are not clinically or informallydiagnosed with one of these diseases, comprises administering one ormore of the dosage forms comprising about 0.6 mgA of colchicine. Thecolchicine in the dosage form can be ultrapure colchicine.

The invention should not be considered limited to these particularconditions for combining the components and it will be understood, basedon this disclosure that the advantageous properties can be achievedthrough other conditions provided the components retain their basicproperties and substantial homogeneity of the blended formulationcomponents of the formulation is otherwise achieved without anysignificant segregation.

The following examples further illustrate the invention but should notbe construed as in any way limiting its scope. In particular, theprocessing conditions are merely exemplary and can be readily varied byone of ordinary skill in the art.

EXAMPLES Example 1 Exemplary Ultrapure Colchicine

As discussed above, ultrapure colchicine with reduced levels ofindividual and total impurities was desired by the inventors forformulation into a new dosage form in order to minimize potentialadverse reactions from the impurities in patients taking the dosage formand to reduce the expense of qualification testing during the approvalprocess for marketing the new dosage form. Batches of conventionalcolchicine were previously obtained from Sanmar Specialty ChemicalsLimited (Berigari, India). Conventional colchicine can be furtherpurified to form ultrapure colchicine meeting the following impurityspecifications:

TABLE 4 Purity Specifications for an exemplary batch of UltrapureColchicine Impurity, Common name Impurity NMT % N-deacetyl-N-formylcolchicine A 0.10 Conformational isomer B 1.0 β-Lumicolchicine C 0.10Colchicoside D 0.10 3-O-demethyl colchicine E 0.10 Total Impurities 2.0

Ultrapure colchicine was prepared to meet the purity specifications inTable 4 as described below.

First, conventional colchicine was obtained from a colchicine chloroformextract. The extract was washed with a mixture of purified water, sodiumhydroxide solution, sodium chloride solution and acetic acid. The washedextract was filtered and the resulting concentrate was distilled in twosteps, first using methanol, and second using ethyl acetate. Theresulting distillate was crystallized. Ethyl acetate was used to isolateand wash the crystallized colchicine, which was then dried, resulting inthe conventional colchicine. This process is also referred to herein asthe “old process”.

Second, the conventional colchicine was then subjected to columnchromatography on neutral alumina using methylene chloride as solvent.The resulting concentrate was distilled using ethyl acetate,crystallized, isolated and washed using ethyl acetate, and dried,resulting in ultrapure colchicine. This method of generatingconventional colchicine, followed by the additional chromatographypurification step is also referred to herein as the “new process”.

The impurity levels of the lot of ultrapure colchicine and two lots ofconventional colchicine were analyzed using the USP30/NF25 ColchicineOfficial Monograph HPLC method (“USP method”) described in Table 2above. The impurity levels are shown in Table 5.

TABLE 5 Impurity Level, % N-Deacetyl-N- Conformational Total formylcolchicine - Isomer - Unidentified Total Colchicine Lot Impurity AImpurity B Impurities Impurities Ultrapure (RD0600164) ND* 0.5 ND* 0.5Conventional-1 (RD060075) 2.1 0.6 ND* 2.7 Conventional 2 (RD060055) 2.20.6 ND* 2.8 *ND—None detected.

Table 5 shows that ultrapure colchicine has fewer impurities than eachof the conventional colchicine lots. Total impurities of the UltrapureLot using the USP method were about 0.5%. On the other hand, totalimpurities of conventional Lots #1 and 2 were about 2.7% and 2.6%,respectively.

Determined impurity levels may differ depending on the test method used.Table 5B contrasts the determined levels of the conformational isomer,Impurity A (N-deacetyl-N-formyl colchicine), and total impurities forthe three colchicine lots using the three methods described in Table 2.The three methods show a maximum absolute variability in the percentdetermined of 0.8% for N-deacetyl-N-formyl colchicine, of 0.5% forconformational isomer, and 0.7% for total impurities.

TABLE 5B Levels of impurities in colchicine lots determined usingmethods of Table 2. N-deacetyl-N-formyl Conformational Isomer colchicineTotal Impurities Lot name Purification UPLC HPLC USP UPLC HPLC USP UPLCHPLC USP (Lot #) Process Method Method Method Method Method MethodMethod Method Method Conventional-1 Old 0.9 0.8 0.6 3.0 2.5 2.2 3.5 2.8(RD060055) Conventional 2 Old 0.9 0.8 0.6 2.7 2.3 2.1 3.2 2.7 (RD060075)Ultrapure New 0.9 1.0 0.5 ND* ND ND 1.1 0.5 (RD0600164) *ND, nonedetected.

Regardless of the testing method used for measuring these impuritylevels, the impurity levels for ultrapure colchicine manufactured usingthe new process remains below the specifications set forth in Table 4.

The ultrapure colchicine of the present invention was prepared to meetthe organic volatile impurity specifications (“residual solvents”) inTable 6 as described below. The residual solvents are determined usingUSP <467> test method. In addition to the known and existing solvents,specifications were also set for residual solvent peaks that were seenin HPLC assay testing as described in Tables 2, 3A, or 3C, whichsolvents are not expected or previously existing for the residualsolvent test methods for colchicine or were not identifiable.

TABLE 6 Specifications for Organic Volatile Impurities Organic volatileNMT Chloroform  100 ppm Methanol 3000 ppm Methylene Chloride  600 ppmEthanol 5000 ppm Ethyl Acetate 6.0% Ethyl Propionate 5000 ppm PropylAcetate 5000 ppm Others  500 ppm each

Example 2 Stable Tablets Comprising Ultrapure Colchicine

Stable colchicine compositions comprising the ultrapure colchicinedescribed in Example 1 were manufactured using the following process.Ultrapure colchicine as described in Example 1 was dissolved in purifiedwater. Pregelatinized starch (Starch® 1500), lactose monohydrate, NF(Fast Flo), and microcrystalline cellulose, NF (Avicel PH101) wereplaced in a 150-liter high shear granulator and mixed. The aqueousultrapure colchicine solution was added to the granulator while mixing.The wet granules were dried in an oven at 50° C. until the loss ondrying of the material was less than 3 wt %. The dried granules weremilled through a Fitzmill equipped with a 1A screen.

The milled granules were charged into a 5 cubic foot Gemco Double ConeBlender and blended with screened sodium starch glycolate, NF(GLYCOLYS®). Then, screened magnesium stearate, NF was added to theblender. Blending was continued and a final tableting blend was made.This final tableting blend was compressed into core tablets. These coretablets were film-coated with OPADRY® II purple and carnauba wax. Thecomposition of the ultrapure colchicine tablets is shown in Table 7.

TABLE 7 Ingredient Amount Per Tablet, mg Ultrapure Colchicine 0.6¹Pregelatinized starch, NF (Starch 1500) 14.0 Lactose Monohydrate, NF(Fast Flo) Varies² Microcrystalline Cellulose, NF (Avicel PH101) 21.6Sodium Starch Glycolate, NF (GLYCOLYS) 4.3 Magnesium Stearate, NF 0.6Total core tablet 100 OPADRY II Purple (#40L10039) 4.0 Carnauba Wax 0.01¹Colchicine amount is shown in units of mgA, adjusted for purity of thelot of colchicine. ²Amount adjusted, depending on the actual amount ofthe colchicine lot added, to maintain an overall core tablet weight of100 mg.

As a comparison, the lot of conventional colchicine designated inExample 1 as “conventional-2” was substituted in place of ultrapurecolchicine in the same tablet formulation shown in Table 7. The sameprocess of making the tablets as described above was used.

The impurities in the tablet comprising the ultrapure colchicine andthat comprising the conventional colchicine were analyzed using the HPLCmethod described in Table 2 above. The impurity levels of bothcolchicine tablets are shown in Table 8.

TABLE 8 Impurity Content, % N-Deacetyl-N- Conformation Total Colchicineformyl colchicine Isomer Unknown Total Product Lot Colchicine LotProcess (Impurity A) (Impurity B) Impurities Impurities A Ultrapure NewND* 1.1 0.1 1.2 B Conventional-2 Old 2.3 1.2 ND* 3.6 *ND—None detected.

It can be seen from Table 8 that the tablet comprising the ultrapurecolchicine has less total impurities than that comprising theconventional colchicine.

The colchicine composition comprising ultrapure colchicine at 6 monthstability time points under conditions of 25° C./60% relative humidityand 40° C./60% relative humidity exhibits impurity content ranges of NotDetected to about 0.1% for Impurity A, less than about 1.0% for totalunknown impurities compared to a colchicine composition comprisingconventional colchicine which exhibits impurity content of greater thanabout 1.5 for Impurity A and greater than about 2.0% for total unknownimpurities when tested under the same conditions.

Table 9 below provides data on impurity levels and stability of impuritylevels of colchicine composition batches manufactured using ultrapureand conventional colchicine, and impurity levels for two lots ofcommercially available COL-PROBENECID® tablets (Watson Laboratories), anFDA-approved combination dosage form comprising colchicine andprobenicid.

TABLE 9 Conformational N-Deacetyl Colchicine Isomer peak purificationConditions of Stability UPLC HPLC UPLC HPLC Material Lot Process StudyMethod Method Method Method COL-PROBENECID ® L6C0395 N/A N/A 0.8 — 2.2 —⁽Probenecid/ L6M1440 N/A N/A 0.8 — 2.5 — Colchicine) Tablets† ColchicineProduct B Old room temp, at release 0.9 1.2 2.8 2.3 Lot process 12 mo 25C./60% RH  0.9 0.9 2.7 2.6 A New room temp, at release 1.0 1.2 ND NDprocess 6 mo 25 C./60% RH 1.0 0.8 ND ND 6 mo 40 C./75% RH 1.0 1.1 ND NDC New room temp, at release 1.0 1.1 ND ND process 6 mo 25 C./60% RH 0.90.9 ND ND 6 mo 40 C./75% RH 1.0 1.1 ND ND D New room temp, at release1.0 1.1 ND ND process 6 mo 25 C./60% RH 1.0 1.0 ND ND 6 mo 40 C./75% RH0.9 1.1 ND ND —, not analyzed; †Commercially available; N/A, notapplicable; ND, none detected.

For comparison, several lots of an FDA-approved colchicine-probenecidcombination dosage form and various unapproved commercial colchicinedosage forms were tested for levels of impurities using the HPLC methodof Table 2. Results are shown in the tables below.

Impurities in FDA-Approved Colchicine/Probenecid Combination Product

Watson Laboratories Colchicine/Probenecid Tablets Impurity L7G1085L7G1085 L7G1087 L7E0808 Conformational 1.0% 1.0% 0.8% 1.0% IsomerN-deacetyl-N-formyl 2.0% 2.0% 1.5% 2.0% colchicine Largest Unknown 0.1%0.1% 0.1% 0.1% Total Impurities 3.1% 3.1% 2.4% 3.2%

Impurities in Unapproved Colchicine Products

West-Ward Vision Impurity 62303A* 63842A 63843A C07003 C07049 C07058 ExpDate Jan-2009 May-2011 May-2011 Jan-2009 Aug-2009 Sep-2009Conformational Isomer 1.1/0.9% 0.9% 0.9% 1.1/0.8% 0.9% 0.9%N-deacetyl-N-formyl colchicine 2.5/2.6% 2.0% 1.8% 1.3/1.3% 2.7% 2.6%Largest Unknown 1.7/1.6% 0.5% 0.3% 0.1/0.1% 0.1% 0.3% Total Impurities5.3/5.3% 3.5% 3.1% 2.5/2.3% 3.8% 4.0% Qualitest Akyma Impurity T105G07AT107G07A T108G07A 3A5246004* Exp Date Jul-2010 Jul-2010 Aug-2010Jan-2008 Conformational 1.0% 0.9% 0.9% 1.1/0.9% Isomer N-deacetyl-N-1.4% 1.3% 1.3% 1.4/1.5% formyl colchicine Largest Unknown 0.3% 0.2% 0.2%0.2/0.1% Total Impurities 2.7% 2.7% 2.6% 2.9/2.5% *Values from twoseparate analyses reported

Summary of Impurities in Marketed Colchicine Products with Batches ofColchicine Tablets Using Ultrapure Colchicine

Marketed Colchicine Product Lots with Products Ultrapure ColchicineImpurity Minimum Maximum Maximum Conformational Isomer 0.8% 1.1% 1.1%N-deacetyl-N-formyl 1.3% 2.7% ND colchicine Largest Unknown 0.1% 1.7%0.3% Total Impurities 2.4% 5.3% 1.4% ND = none detected

The total impurities found in ultrapure colchicine and colchicineproducts comprising ultrapure colchicine are significantly lowercompared to the approved and unapproved, marketed colchicine products.In addition, the maximum total impurities value observed by testing 14approved or unapproved marketed products was 5.3%; while the maximumvalue seen to date in inventive ultrapure colchicine product batches was1.4%. This represents a 75% reduction in total impurities.

Of particular significance, the level of the known impurity,N-deacetyl-N-formyl-colchicine (Impurity A, also known as Gloriosine)has been reduced from levels exceeding 2% to levels to undetectablelevels that comply with the ICH Q3A(R2) qualification threshold of 0.15%for an active agent. Gloriosine is tumorigenic and has been studied asan anti-cancer agent. Purification of conventional colchicine to obtainultrapure colchicine has effectively reduced all individual impuritylevels in the colchicine to substantially reduce exposure of patients tothis tumorigenic impurity.

Example 3 Therapeutic Effects of Ultrapure Colchicine Formulation

The therapeutic effect of the ultrapure colchicine formulationcontaining 0.6 mgA of colchicine obtained in Example 2 is evaluated in aclinical study that is a multicenter, randomized, double-blind,placebo-controlled, parallel group, 1-week, dose comparison studydesigned to evaluate the efficacy of ultrapure colchicine in treating anacute gout attack (acute gouty arthritic attack) in patients with acutegout. A sufficient number of patients are screened to enroll andrandomize 300 patients (100 patients per treatment group) who meet thecriteria of the American College of Rheumatology (ACR) for acutearthritis of gout. The primary objective of the study is to demonstratethe efficacy of colchicine in an acute gout attack (gouty flare) basedon pain reduction after 24 hours as a measure of response. Secondaryobjectives of the study are to compare low-dose and standard-dose dosingregimens of colchicine with respect to pain, time to response andcomplete pain relief, interference with sleep, and signs and symptoms ofinflammation and to determine the safety of colchicine when administeredin the two different dosing regimens.

Description of Study

The study will consist of three distinct phases and the number of visitsto the study clinic will vary depending on conditions pertaining to anindividual patient's acute gout flare experienced in the study asdescribed below. The Pre-Flare Phase will consist of up to two visits tothe study clinic (with additional interim visits for clinical laboratorytesting every 3 months until acute gout flare onset): Visit 1(Screening) and Visit 2 (Randomization). The Flare Phase will notinclude a visit to the study clinic. The Post-Flare Phase will consistof up to three visits to the study clinic: Visit 3 (as soon as possible[ASAP] up to 48 hours post-flare onset; if a patient cannot completeVisit 3 in the first 48 hours post-flare onset, Visit 3 will be waivedand the patient should return to the clinic for Visit 4), Visit 4 (>48to 96 hours post-flare onset in patients who took at least one dose ofstudy drug and in patients who did not qualify for treatment with studydrug during the Flare Phase but did not complete Visit 3), and Visit 5(7 days post-flare onset to be conducted in patients who took at leastone dose of study drug and whose acute gout flare was still ongoing atVisit 4). For those patients in whom the acute gout flare is notresolved at Visit 5 (based on the judgment of the Investigator) or inwhom there is an unresolved AE or clinically significanttreatment-emergent laboratory abnormality, there will be one additionalfollow-up visit 14 days post-flare onset (Visit 6).

When a patient develops an acute gout flare, the patient will call thetrained personnel at the Gout Flare Call Center (available 24 hours/daythroughout the duration of the study). The patient will be queriedregarding any changes in his/her medical health and concomitantmedication use since the time of randomization. In order to establish ifthe patient's acute gout flare will be eligible for treatment with studydrug, a standardized questionnaire will be used to document that thepatient has all of the following signs/symptoms of the affectedjoint(s): swelling, erythema, marked tenderness, and pain. In addition,a patient must have at least one of the following: rapid onset ofmaximum pain within the prior 4 to 12 hours, decreased range of motionin the joint, warmth, or other symptom similar to a prior gout flare.Patients will be asked to rate the pain severity for each joint affectedby the acute gout flare by using a study diary. Patients must have atleast one joint affected by an acute gout flare with a pain assessmentof ≧4 on the PI-NRS at the onset of the acute gout flare during theFlare Phase prior to taking study drug. If signs and symptoms of anacute gout flare are confirmed and the gout flare is considered eligiblefor treatment with study drug, the patient will also be instructed tobegin taking study drug and to continue completing the patient diary.Patients will be instructed to stop taking study drug and to call theinvestigational site if at any time they experience a severegastrointestinal event while taking study drug. The Gout Flare CallCenter will call the patient in 24 hours from the onset of the acutegout flare to ensure that the patient has completed the patient diary,including assessments for pain at 24 hours.

In the event that the Gout Flare Call Center determines that the patientdoes not qualify for treatment with study drug, the patient may be askedto call back in 1 hour for re-assessment. Patients who do not qualifyfor treatment with study drug may seek alternative therapy withoutprejudice to further study participation should another acute gout flareoccur.

The Gout Flare Call Center will contact patients on a monthly basisbeginning 1 month after randomization to study drug. These contacts willcontinue until the patient has an acute gout flare or study completion,whichever occurs first. The purpose of these contacts is to re-educatepatients about study participation.

Post-Flare Phase:

Visit 3: After developing an acute gout flare, whether eligible fortreatment with study drug or not, all patients will return to the clinicas soon as possible after acute gout flare onset for clinicalassessments. If a patient cannot complete Visit 3 in the first 48 hourspost-flare onset, Visit 3 will be waived and the patient should returnto the clinic for Visit 4. For patients whose acute gout flare wasdeemed eligible for treatment with study drug and at least one dose ofstudy drug was taken, Investigators will examine the patient andcomplete clinical assessments, patient diaries will be collected, thepatient will be queried for concomitant medication use and AdverseEvents (AEs), and the study drug blister pack will be collected. Forpatients whose acute gout flare was deemed not eligible for treatmentwith study drug by the Gout Flare Call Center, continued eligibility forthe study will be confirmed by the Investigator based on review of theinclusion and exclusion criteria as well as the responses to thestandardized questionnaire and examination of the patient. Patientdiaries will be collected and patients will be queried for concomitantmedication use and AEs.

Visit 4: The second Post-Flare Phase visit is to be conducted >48 to 96hours following acute gout flare onset. It will take place in patientswho took at least one dose of study drug and also in those patients whodid not qualify for treatment with study drug during the Flare Phase butdid not complete Visit 3. For patients whose acute gout flare was deemedeligible for treatment with study drug and at least one dose of studydrug was taken, Investigators will examine the patient and completeclinical assessments, patient diaries will be collected, patients willbe queried for concomitant medication use and AEs, and the study drugblister pack will be collected (if not previously collected). Patientswho took at least one dose of study drug and whose acute gout flare isstill ongoing at Visit 4 will return to the clinic for a final visit(Visit 5) 7 days post-flare onset; however, if their acute gout flare isresolved at Visit 4, final study assessments, including collection ofsamples for laboratory safety and a complete physical examination, willbe performed at Visit 4. For patients whose acute gout flare was deemednot eligible for treatment with study drug by the Gout Flare Call Centerwho did not have a Visit 3, continued eligibility for the study will beconfirmed by the Investigator based on review of the inclusion andexclusion criteria as well as the responses to the standardizedquestionnaire and examination of the patient. Patient diaries will becollected and patients will be queried for concomitant medication useand AEs.

Visit 5/Early Termination: The final visit for the study will take place7 days after the acute gout flare onset in patients who took at leastone dose of study drug and whose acute gout flare was still ongoing atVisit 4. Patients will be examined and clinical assessments will bemade. A complete physical examination will be conducted. Samples forclinical laboratory testing will be collected. Patient diaries will becollected and patients will be queried for concomitant medication useand AEs. The study drug blister pack will be collected (if notpreviously collected).

Visit 6/Follow-up: For those patients in whom the acute gout flare isnot resolved at Visit 5 (based on the judgment of the Investigator) orin whom there is an unresolved AE or clinically significanttreatment-emergent laboratory abnormality, there will be one additionalfollow-up visit 14 days post-flare onset (Visit 6).

For inclusion in the study, a patient must be 18 years of age or older,must present with a confirmed diagnosis of gout consistent with thecriteria of the ACR, and must have experienced ≧2 acute gouty arthriticattacks in the 12 months prior to randomization. Patients onurate-lowering therapy must be on a stable dose and schedule with nochanges in therapy for 4 weeks prior to randomization and expected toremain on a stable regimen during study participation.

Patients with acute polyarticular gout (>4 joints); taking colchicineroutinely; with a known hypersensitivity to colchicine; with a historyof myocardial infarction, unstable angina, cerebrovascular events, orcoronary artery bypass grafting; with active myeloid leukemia,obstructive gastrointestinal cancer, or metastatic cancer; with chronicrenal dysfunction, with chronic hepatic dysfunction are excluded fromthe study. Patients using systemic corticosteroid, cyclosporine,adalimumab, etanercept, infliximab, anakinra, abatacept, mycophenolate,azathioprine, or chronic use of non-steroidal anti-inflammatory drugs(NSAIDs), acetaminophen, tramadol, and other analgesics such as opiatesat screening are also be excluded.

The three treatment groups in this study are two ultrapure colchicinetablets (1.2 mgA) administered at the onset of an acute gout attackfollowed by one tablet (0.6 mgA) after one hour and one placebo tabletevery hour thereafter for 5 hours (the “low” dose regimen); twoultrapure colchicine tablets (1.2 mgA) administered at the onset of anacute gout attack followed by one tablet (0.6 mgA) every hour for six(6) hours (the “standard” dose regimen); or two placebo tablets at theonset of an acute gout attack followed by one placebo capsule every hourthereafter for 6 hours (the “placebo” regimen).

Efficacy assessment will be made based on patient and Investigatorinputs. Patients will record pain severity and sleep interference on astudy diary. The severity of pain for each joint affected by the acutegouty arthritic attack will be rated on an 11-point pain intensitynumerical rating scale (PI-NRS) that ranges from 0 (“no pain”) to 10(“worst possible pain”). Recordings are to be made prior to each dose ofstudy drug, i.e., pre-treatment with study drug and for the first 8hours following start of treatment, and every 8 hours thereafter (whileawake) until symptoms disappear or 72 hours have passed since the firstdose of study drug was taken, whichever occurs first. The patient's painassessment of each affected joint as reported by the patient atpre-treatment with study drug and at 24 hours post-start of study drugwill be documented by a central Study Center to be used in the event thepatient fails to provide data on his/her study diary for either of thesetwo key time points. In the morning upon awakening, the patient is torate sleep interference due to the acute gout flare in the study diaryon an 11-point scale that ranges from 0 (“pain did not interfere withsleep”) to 10 (“pain completely interfered; patient was unable tosleep”). Investigators will provide clinical assessments in the clinicat all Post-Flare Phase study visits, and at medically necessaryintervening visits. For these assessments, each of the patient's jointsaffected by the acute gouty arthritic attack will be examined and signsand symptoms of inflammation will be rated (erythema [absent, present,or not assessable], swelling [0, “none” to 3, “severe”], and tendernessto touch [0, “none” to 3, “severe”]). At the final clinic visit, theInvestigator will also provide a global assessment of response totreatment ranging from 0 (“excellent”) to 4 (“none”).

The primary efficacy variable is response to treatment in the targetjoint, based on patient self-assessment of pain at 24 hours post-dose.The target joint is identified during data analysis as that jointaffected by the acute gout flare with the highest baseline pain score onthe patient diary. Ties among maximum joint scores for an individual areresolved by random selection. A responder is one who provides both apre-treatment and valid 24-hour pain score and achieves a ≧50% reductionin pain score at the 24-hour post-dose assessment relative to thepre-treatment score and does not use rescue medication. Patients who userescue medication, discontinue prior to the 24-hour post-doseassessment, or do not achieve a ≧50% reduction in pain score at the 24hour post-dose assessment relative to the pre-treatment score are deemednon-responders.

The secondary efficacy variables are assessments of magnitude of painreduction, time to response, time to complete pain relief, andinterference with sleep as recorded on patient diaries by the patient.Signs and symptoms of inflammation per Investigator's clinicalassessments of the target joint are evaluated. Time to drop out (use ofrescue medications) is also evaluated. Investigator global assessment ofresponse to treatment is assessed.

The primary efficacy analysis will be based on an Intent-to-Treat (ITT)population, defined as all patients who were randomized, contacted theGout Flare Call Center, took at least one dose of study drug, and hadone subsequent contact. The Per Protocol (PP) population is defined asthe subset of the ITT population confirmed by the Investigator ascontinuing to meet all major inclusion and exclusion criteria andinitiating treatment within 12 hours of the onset of the acute goutflare. Analyses will also be made on an Evaluable patient populationwith an Evaluable patient defined as one who, in addition to beingincluded in the PP population, completed the randomized treatmentcourse. The ITT population will be used for the evaluation of safety.

Statistical tests for efficacy analysis are two-tailed with an alphasignificance level of 0.05.

For primary efficacy analysis, the number of responders in thestandard-dose colchicine group and the placebo group, as defined for theprimary efficacy variable, will be compared using the Mantel-Haenszelchi-square test stratified on study site. The comparison of thestandard-dose colchicine and placebo groups of the ITT population is theprimary comparison of interest. The sensitivity to alternate definitionsof response (based both on magnitude of reduction from baseline as wellas time point) will be evaluated as secondary endpoints. As additionalsensitivity analyses, these tests will also be repeated for the PP andEvaluable populations if the sample sizes differ from the overall ITTpopulation by more than 10%.

For secondary efficacy analysis, the number of responders in thelow-dose colchicine group will be compared to placebo and also tostandard-dose colchicine using the Mantel-Haenszel chi-square teststratified on study site. Change in pain intensity, interference withsleep, time to 50% reduction in pain, and time to complete pain reliefwill be analyzed as continuous variables using analysis of covariancewith study site, treatment group, and site by treatment interaction asindependent variables for change in pain intensity and interference withsleep, with baseline score as a covariate. For the Investigator'sclinical assessment of inflammation (erythema, swelling, and tendernessto touch) and Investigator's global assessment of response to treatment,the treatment groups will be compared using the Mantel-Haenszelchi-square test stratified on study site.

Safety assessments will be made based on patient and Investigatorinputs. Patients will be initially screened in the clinic for inclusionby review of medical history and concomitant medication use, physicalexamination, measurement of vital signs (oral temperature, sittingradial or brachial pulse rate, respiratory rate, and sitting bloodpressure), body weight, and clinical laboratory testing (serumbiochemistry, complete blood count, and urinalysis). Afterrandomization, clinical laboratory tests, concomitant medication use,medical history and current complaints (AE) will be reviewed every 3months until an acute gout flare occurs in order to ensure continuedeligibility. Compliance with key inclusion/exclusion criteria (based onintervening medical history and concomitant medication use) will be reconfirmed by the Gout Flare Call Center prior to authorizing the startof study drug. Following the start of study drug, patients will recordany severe gastrointestinal AEs on their diaries and these will berecorded in the Case Report Form (CRF) at each clinic visit. Fullphysical examinations and clinical laboratory testing will be conductedat the final clinic visit (Visit 4 or Visit 5). Vital signs and bodyweight will be measured at each Post Flare visit (Visit 3 and 4 or 5).For those patients in whom there is an unresolved AE or clinicallysignificant treatment emergent laboratory abnormality, there will be oneadditional follow up visit 14 days post flare onset (Visit 6).

Safety analysis will be performed by coding adverse events using astandardized medical dictionary and the incidence summarized bytreatment group; tabulations will be prepared of all AEs as well as byrelationship and by severity. Adverse events resulting in terminationand events meeting regulatory criteria for seriousness will also betabulated separately. Descriptive statistics (mean, median, standarddeviation, and range) of clinical laboratory testing results and vitalsign measurements will be generated for each treatment group and changefrom the most recent value prior to onset of the acute gout flarecalculated; no inferential testing will be performed. Treatment emergentabnormalities on physical examination will be tabulated and listed bytreatment group. By patient listings of all safety data and concomitantmedication use will be generated.

Study Results

The following data were obtained in general accordance with the aboveprotocol.

Out of 813 patients screened, 575 were randomized to treatment with 185patients having a gouty flare and receiving study drug. Theintent-to-treat population consisted of 184 patients (52 receivedstandard dose, 74 received low dose and 58 received placebo).

Number of Responders Based on Target Joint Pain Score at 24 Hours PostFirst Dose

Colchicine Dose Placebo Odds Ratio (95% Confidence Intervals) Low (N =74) High (N = 52) (N = 58) Low vs. High vs. High vs. N (%) N (%) N (%)Placebo Placebo Low 28 (37.8) 17 (32.7) 9 (15.5) 3.31 (1.41, 7.77) 2.64(1.06, 6.62) 0.80 (0.38, 1.68) P = 0.0046 P = 0.0343 P = 0.5529

Cumulative Distribution of Degree of Percent Improvement for TargetJoint Pain Score at 24 Hours Post First Dose

Colchicine Dose % Improvement High (N = 52) Low (N = 74) Placebo (N =58) >=0%  52 (100.0%)  74 (100.0%)  58 (100.0%) >=10% 32 (61.5%) 47(63.5%) 24 (41.4%) >=20% 29 (55.8%) 45 (60.8%) 21 (36.2%) >=30% 21(40.4%) 39 (52.7%) 17 (29.3%) >=40% 21 (40.4%) 36 (48.6%) 14(24.1%) >=50% 19 (36.5%) 30 (40.5%) 10 (17.2%) >=60% 15 (28.8%) 24(32.4%)  7 (12.1%) >=70% 10 (19.2%) 20 (27.0%) 4 (6.9%) >=80%  9 (17.3%)15 (20.3%) 3 (5.2%) >=90%  6 (11.5%)  9 (12.2%) 2 (3.4%) >=100%  6(11.5%)  8 (10.8%) 2 (3.4%)

Treatment Response Based on at Least a 2-Unit Reduction in Target JointPain Score at 24 Hours and 32 Hours Post First Dose

Number (%) of Responders Treatment Comparisons Colchicine Dose (OddsRatio and 95% CI)¹ Hours Post High Low Placebo High vs. Low vs. High vs.First Dose (N = 52) (N = 74) (N = 58) Placebo Placebo Low 24 18 (34.6)32 (43.2) 10 (17.2) 2.54 (1.04, 6.18) 3.66 (1.61, 8.32) 0.69 (0.33,1.45) p = 0.0368 p = 0.0015 p = 0.3298 32 20 (38.5) 34 (45.9) 10 (17.2)3.00 (1.24, 7.24) 4.08 (1.80, 9.27) 0.74 (0.36, 1.51) p = 0.0126 p =0.0005 p = 0.4033 ¹The p-value is from the unstratified Pearsonchi-square test.Target Joint Pain at Baseline, 24 Hours and 32 Hours Post First Dose,and Change from Baseline (LOCF)—ITT Population

Colchicine Dose Treatment Comparisons¹ High Low Placebo High vs. Low vs.High vs. Time Point Statistic (N = 52) (N = 74) (N = 58) Placebo PlaceboLow 24 Hours Post First Dose Baseline Mean (SD)  6.9 (1.59)  6.9 (1.72) 6.8 (1.44) −1.3 −1.5 0.2 Median  7.0 (4, 10)  7.0 (4, 10)  7.0 (4, 10)p = 0.0145 p = 0.0055 p = 0.7540 (Mix, Max) Change Mean (SD) −2.0 (2.93)−2.2 (3.46) −0.7 (2.77) Median −2.0 (−9, 4) −2.0 (−9, 5) −0.0 (−8, 4)(Mix, Max) 32 Hours Post First Dose Baseline Mean (SD)  6.9 (1.59)  6.9(1.72)  6.8 (1.44) −1.6 −1.6 0.1 Median  7.0 (4, 10)  7.0 (4, 10)  7.0(4, 10) p = 0.0057 p = 0.0038 p = 0.9238 (Mix, Max) Change Mean (SD)−2.3 (3.05) −2.4 (3.59) −0.7 (2.95) Median −2.0 (−9, 3) −2.5 (−9, 5) 0.0 (−8, 4) (Mix, Max) ²Tabled values are the difference betweentreatment groups mean change from baseline and p-value from ANCOVA withtreatment group as the independent variable and baseline score as thecovariate.

Total Pain Relief (TOTPAR) Based on all Target Joint Pain Scores

Colchicine Dose High Low Placebo Time Point Statistic (N = 52) (N = 74)(N = 58) Hour 24 n 51¹ 74 58 Mean (SD) 20.9 (48.42) 30.5 (61.44)  9.5(45.87) Median (Mix, Max) 11.5 (−102, 135) 23.0 (−112, 185)  7.3 (−90,142) Hour 32 n 51 74 58 Mean (SD) 31.9 (63.83) 45.5 (82.05) 12.2 (59.88)Median (Mix, Max) 27.5 (−102, 185) 34.1 (−128, 257)  7.3 (−114, 142)¹Patient 1026-1005 did not have a diary and the 24-hour call to the CallCenter was 27 hours after the initial call. As indicated in the SAP, theCall Center pain score was not eligible for substitution for the missingdiary. This patient has been excluded from the TOTPAR summary.

Number (%) of Patients Using Rescue Medication Up to and Including the24-Hour Post First Dose Assessment

Colchicine Dose High Low Placebo Treatment Comparison (N = 52) (N = 74)(N = 58) (Odds Ratio and 95% CI) n (%) n (%) n (%) High vs. Placebo Lowvs. Placebo High vs. Low 18 (34.6) 23 (31.1) 29 (50.0%) 0.53 (0.25,1.14) 0.45 (0.22, 0.92) 1.17 (0.55, 2.50) p = 0.1034 p = 0.0273 p =0.6768Change from Baseline in Target Joint Pain Scores at 24 Hours Post FirstDose with Interval of Time of Dose Relative to Flare Onset as Covariate(LOCF)—ITT Population

Colchicine Dose Treatment Comparisons¹ High Low Placebo High vs. Low vs.High vs. Statistic (N = 52) (N = 74) (N = 58) Placebo Placebo Low EarlyTreatment Start (within 4 hours) Baseline Mean (SD)  6.9 (1.59)  6.9(1.72)  6.8 (1.44) −1.3 −1.5 0.2 Median  7.0 (4, 10)  7.0 (4, 10)  7.0(4, 10) p = 0.0145 p = 0.0055 p = 0.7540 (Mix, Max) Change Mean (SD)−2.0 (2.93) −2.2 (3.46) −0.7 (2.77) Median −2.0 (−9, 4) −2.0 (−9, 5)−0.0 (−8, 4) (Mix, Max) Late Treatment Start (after 4 hours) BaselineMean (SD)  6.9 (1.59)  6.9 (1.72)  6.8 (1.44) −1.6 −1.6 0.1 Median  7.0(4, 10)  7.0 (4, 10)  7.0 (4, 10) p = 0.0057 p = 0.0038 p = 0.9238 (Mix,Max) Change Mean (SD) −2.3 (3.05) −2.4 (3.59) −0.7 (2.95) Median −2.0(−9, 3) −2.5 (−9, 5)  0.0 (−8, 4) (Mix, Max) ¹Patient 1016-1013 wasmissing a flare onset time and Patients 1002-1007, 1018-1008, 1006-1013,1009-1011, 1010-1005, 1010-1010, 1026-1002, 1026-1007, 1064-1007, and1068-1022 appear to have taken the first dose of study medication priorto flare onset. ²Tabled values are the difference between treatmentgroups mean change from baseline and p-value from ANCOVA with treatmentgroup as the independent variable and baseline score as the covariate.

Overall Summary of Treatment Emergent Adverse Events—Safety Population

Colchicine Dose High Low Placebo (N = 52) (N = 74) (N = 59) n (%) n (%)n (%) Total Number of TEAEs¹ 85  34  27  Number (%) of Patients with at40 (76.9) 27 (36.5) 16 (27.1)  Least One TEAE Number (%) of Patientswith at 15 (28.8) 19 (25.7) 9 (15.3) Least One Mild TEAE Number (%) ofPatients with at 15 (28.8)  8 (10.8) 6 (10.2) Least One Moderate TEAENumber (%) of Patients with at 10 (19.2) 0 1 (1.7)  Least One SevereTEAE Number (%) of Patients with a 0 0 0 TEAE Discontinuing Study Number(%) of Patients with a 0 0 0 Treatment Emergent SAE ¹Patients reportingmore than one adverse event are only counted once for a given event.

Number (%) of Patients with at Least One Treatment-EmergentGastrointestinal Adverse Event Recorded on the Diary or the CRF SafetyPopulation

Colchicine Dose Standard Low Placebo (N = 52) (N = 74) (N = 59) Methodof Capture All Severe All Severe All Severe Captured on Adverse 40(76.9)² 10 (19.2) 19 (25.7) 0 12 (20.3) 0 Event CRF¹ Captured on Patient48 (92.3)² 13 (25.0) 32 (43.2)³ 3 (4.1) 15 (25.4) 2 (3.4) Diary Capturedon Patient 49 (94.2)² 18 (34.6) 33 (44.6) 3 (4.1) 16 (27.1) 2 (3.4)Diary or Adverse Event CRF ¹Gastrointestinal adverse events captured onthe AE CRF include the MedDRA preferred terms of “diarrhoea”, “nausea”,“vomiting”, “abdominal pain”, or “abdominal pain lower”, “abdominal painupper”, “abdominal discomfort”, or “dyspepsia”. ²Statisticallysignificantly different from placebo and from Low-dose colchicine (95%CI of odds ratio does not include “1”). ³Statistically significantlydifferent from placebo (95% CI of odds ratio does not include “1”).Number (%) of Patients with at Least One Severe TEAE in any TreatmentGroup—Safety Population

Odds Ratio Colchicine Dose (95% Confidence High Low All ColchicinePlacebo High Low High MedDRA System Organ Class (N = 52) (N = 74) (N =126) (N = 59) vs. vs. vs. MedDRA Preferred Term n (%) n (%) n (%) n (%)Placebo Placebo Low Number of Patients with at Least One 10 (19.2) 0 10(7.9) 1 (1.7) 13.8 — — Severe TEAE (1.7, 112) Gastrointestinal Disorders10 (19.2) 0 10 (7.9) 0 — — — Diarrhea 10 (19.2) 0 10 (7.9) 0 — — —Melaena  1 (1.9) 0  1 (0.8) 0 — — — Nausea  1 (1.9) 0  1 (0.8) 0 — — —Metabolism and Nutrition Disorders  0 0  0 1 (1.7) — — — Gout  0 0  0 1(1.7) — — — Musculoskeletal and Connective  1 (1.9) 0  1 (0.8) 0 — — —Tissue Disorders Pain in Extremity  1 (1.9) 0  1 (0.8) 0 — — —

Number (%) of Patients with at Least One Drug-Related Treatment EmergentAdverse Events with an Incidence of ≧2% of Patients in any TreatmentGroup

Odds Ratio Colchicine Dose Placebo (95% Confidence Intervals) MedDRASystem Organ Class High (N = 52) Low (N = 74) (N = 59) High vs. Low vs.High vs. MedDRA Preferred Term n (%) n (%) n (%) Placebo Placebo LowNumber of Patients with at Least 38 (73.1) 21 (28.4) 14 (23.7)  8.7 1.36.9 One Drug-Related TEAE (3.7, 20.6) (0.6, 2.8) (3.1, 15.2)Gastro-intestinal Disorders 38 (73.1) 18 (24.3) 11 (18.6) 11.8 1.4 8.4(4.8, 29.0) (0.6, 3.3) (3.8, 19.0) Diarrhea 38 (73.1) 16 (21.6)  8(13.6) 17.3 1.8 9.8 (6.6, 45.4) (0.7, 4.4) (4.3, 22.5) Nausea  7 (13.5)3 (4.1) 3 (5.1)  2.9 0.8 3.7 (0.7, 11.9) (0.2, 4.1) (0.9, 15.0) Vomiting 8 (15.4) 0 0 — — —

As shown in the above tables, standard dose colchicine produced ≧50%pain reduction at 24 hrs without pain rescue in a greater proportion ofpatients than did placebo (32.7% vs. 15.5%, p=0.0343; odds ratio 2.64(95% CI, 1.06, 6.62), and more gastrointestinal side effects thanplacebo (73.1% vs. 18.6%, odds ratio 11.8 {95% CI, 4.8, 29.0}), inparticular more diarrhea than placebo (73.1% vs. 13.6%, odds ratio 17.3{95% CI, 6.6, 45.4}). Low dose colchicine also produced ≧50% painreduction at 24 hrs without pain rescue in a greater proportion ofpatients than did placebo (37.8% vs. 15.5%, p=0.0046; odds ratio 3.31(95% CI, 1.41, 7.77)), and more gastrointestinal side effects thanplacebo (24.3% vs. 18.6%, odds ratio 1.4 {95% CI, 0.7 to 11.9}), but didnot significantly differ from placebo with respect to diarrhea (21.6%vs. 13.6%, odds ratio 1.8 {95% CI, 0.7 to 4.4}). Severe diarrheaoccurred in 19.2% of patients taking high-dose colchicine while notoccurring in the low-dose colchicine group. Vomiting occurred in 15.4%of patients taking high-dose colchicine while not occurring in thelow-dose colchicine group.

Based on the primary efficacy variable of ≧50% pain reduction at 24 hrswithout pain rescue, the proportion of responders to the standard doseand the low dose colchicine regimens was not significantly different(p=0.5529). The odds ratio for being a responder to standard dosecolchicine vs. being a responder to low dose colchicine was 0.80 (95%CI, 0.38, 1.68). The proportion of patients rescued prior to 24 hoursfor the standard dose, low dose and placebo were 34.6%, 28.4% and 48.3%,respectively.

FIG. 1 summarizes efficacy data from the trial. FIG. 1 shows thefraction of all patients improved at 24 hrs post-first dose, regardlessof pain rescue, as a function of the percent improvement in pain foreach of the three treatment methods (standard dose, low dose, placebo).For example, 49% of patients taking the low dose achieved at least 40%relief compared to 24% of the patients on placebo.

Standard dose oral colchicine was established to be effective, butburdened by significant diarrhea. In contrast, although low dosecolchicine was not significantly different from standard dose colchicinein efficacy, low dose colchicine was not significantly different fromplacebo with respect to diarrhea. This trial provides a new evidencebasis for acute gout treatment, specifically supporting the unexpectedsuperiority of a low dose colchicine dosing regimen of 2 tablets of 0.6mg followed in 1 hour by 1 tablet. The higher standard dose colchicinedosing regimen did not improve patient outcome, but did increase adverseevents.

Example 4 Pharmacokinetic Study in Healthy Adults of Single Vs. MultipleOral Doses of Colchicine Tablets

This study was a single-center, open-label, single-sequence, two-periodstudy to evaluate the pharmacokinetic profile of colchicine followingsingle and multiple oral doses of colchicine tablets, 0.6 mg, in healthyvolunteers.

In Period 1, study subjects received a 0.6-mg dose of colchicine afteran overnight fast of at least 10 hours. In Period 2, subjects received a0.6-mg dose of colchicine in the morning and the evening (approximately12 hours later) for 10 days (steady state regimen). Subjects received alight breakfast served 60 minutes following dose administration in themorning and the evening dose was administered 90 minutes after anevening meal on Days 15 through 24 only. On Day 25, the colchicine dosewas administered after an overnight fast of at least 10 hours and lunchwas served 4 hours post-dose. Study periods were separated by a 14-daywashout. Following the single dose and the last dose of the multipledose regimen (beginning on the mornings of Day 1 and Day 25,respectively), blood samples were collected (6 mL each) from eachsubject within 1 hour prior to dosing and after dose administration atstudy hours 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 24 (while confined)and 36, 48, 72, and 96 (on an outpatient basis). Plasma concentrationsof colchicine and its metabolites were determined using validatedLC/MS-MS methods.

Thirteen healthy, non-smoking subjects with a mean age of 25.5 years(range 19 to 38 years) and within 15% of ideal body weight were enrolledin this study. All subjects completed both dosing periods according toprotocol.

After a single dose, plasma concentrations are no longer quantifiable 24hours post-dose in all but 1 subject. After the last dose of the steadystate regimen, concentrations remained quantifiable for 48 to 72 hours.Review of individual subject data shows that no subject experienced asecondary colchicine peak, either following a single dose or uponmultiple dosing.

All 2-O-demethylcolchicine (2-DMC) concentrations were below the levelof quantitation (LOQ, 0.2 ng/mL) and only one sample from 1 subject (of13 subjects) had a detectable 3-O-demethylcolchciine (3-DMC)concentration which was near the level of quantitation. Therefore,metabolites are not discussed further.

In healthy adults, colchicine appears to be readily absorbed when givenorally, reaching a mean maximum plasma concentration of 2.5 ng/mL in 1.5hours after a single dose. The drug is distributed widely, with anapparent volume of distribution of 540 L, greatly exceeding total bodywater. The elimination half-life as calculated following a single oraldose is approximately 5 hours. Levels were not detectable by 24 hourspost-dose and this is therefore not an accurate estimate.Pharmacokinetic parameter values are summarized in the table below.

Review of trough plasma concentrations indicates that steady state wasattained by approximately the eighth day of dosing for most subjects.Colchicine may have a diurnal variation reflected in the observed Cminconcentrations at steady state. Cmin concentrations prior to the morningdose are approximately 12% higher than the Cmin concentrations prior tothe evening dose (Day 23 and Day 24). The mean Cmin concentrationobserved on Day 25 was 0.907 ng/mL.

Colchicine accumulated following administration of multiple doses to anextent greater than expected. Exposure was nearly two-fold higher(approximately 1.7 based on AUC [Day 25 AUC_(0-τ)/Day 1 AUC_(0-∞)] andapproximately 1.5 based on Cmax [Day 25 C_(max)/Day 1 C_(max)]). Thisobservation could be attributable to an underestimation of AUC∞following a single dose. With the higher plasma levels that occur withrepeated dosing, a longer terminal elimination half life is apparent,26.6 hours. Pharmacokinetic parameter values are summarized in thetables below.

TABLE 10 Colchicine Pharmacokinetic Parameter Values FollowingAdministration of A Single Oral Dose of Colchicine 0.6 mg in HealthyAdults AUC_(0-t) AUC_(0-inf) C_(max) T_(max) Kel T_(1/2) (pg-hr/mL)(pg-hr/mL) (pg/mL) (hr) (1/hr) (hr) N 13 13 13 13 13 13 MEAN 10508.5412281.90 2470.77 1.50 0.1829 4.95 STDEV 3544.82 4423.34 706.98 0.540.0592 4.43 % CV 33.73 36.02 28.61 36.00 32.39 89.54 MEDIAN 10560.9011451.45 2714.00 1.50 0.1992 3.48 MIN 4812.88 7252.66 1584.00 1.000.0359 2.84 MAX 18128.65 23838.48 3977.00 3.00 0.2443 19.29

TABLE 11 Colchicine Pharmacokinetic Parameter Values FollowingAdministration of Multiple (b.i.d.) Oral Doses of Colchicine 0.6 mg inHealthy Adults AUC_(0-t) AUC_(0-τ) AUC_(0-inf) C_(max) C_(min) C_(ave)T_(max) Kel T_(1/2) (pg-hr/mL) (pg-hr/mL) (pg-hr/mL) (pg/mL) (pg/mL)(pg/mL) (hr) (1/hr) (hr) N 13 13 13 13 13 13 13 13 13 MEAN 43576.9629056.23 54198.77 3553.15 906.51 1210.68 1.31 0.03 26.60 STDEV 9333.264531.30 9214.54 843.45 152.19 188.80 0.60 0.00 4.33 % CV 21.42 15.5917.00 23.74 16.79 15.59 45.61 16.34 16.26 MEDIAN 41925.10 28452.1554113.43 3734.00 903.50 1185.51 1.00 0.03 26.51 MIN 29328.78 20791.9837599.76 1977.00 636.23 866.33 0.50 0.02 20.82 MAX 58265.35 36083.9567944.65 4957.00 1149.67 1503.50 3.00 0.03 33.65

TABLE 12 Mean (% CV) Colchicine Pharmacokinetic Parameter ValuesFollowing Administration of Single and Multiple (b.i.d.) Oral Doses ofColchicine 0.6 mg in Healthy Adults Vd/F (L) CL/F (L/hr) Colchicine0.6-mg Single Dose (N = 13) Day 1 341 (54.4) 54.1 (31.0) Colchicine 0.6mg b.i.d. × 10 days Day 25 1150 (18.73) 30.3 (19.0) CL = Dose/AUC_(0-t)(Calculated from mean values) Vd = CL/Ke (Calculated from mean values)

In the above table, the parameter CL/F denotes the apparent total bodyclearance after administration, calculated as Total Dose/TotalAUC0-_(tau); and V_(d)/F denotes the apparent total volume ofdistribution after administration, calculated as Total Dose/(TotalAUC_(∞)×K_(el)).

Example 5 Pharmacokinetic Study in Healthy Adults of Low Dose Acute GoutRegimen: 1.8 mg Over 2 Hours

This study was a single-center, single-period, open-labelpharmacokinetic study conducted in healthy subjects under fastingconditions. It was designed to characterize the pharmacokinetic profileof a low-dose regimen of colchicine (1.8 mg over 2 hours) used as one ofthe treatment arms in the randomized, controlled trial in patients withan acute gout flare discussed above.

Thirteen healthy, non-smoking subjects with a mean age of 29.3 years(range 20 to 49 years) and within 15% of ideal body weight were enrolledin this study. Subjects received 2×0.6 mg tablets initially followed by1×0.6 mg tablet 1 hour later. Blood samples for measurement ofcolchicine plasma concentrations and metabolites were collected(relative to the first dose of study drug) at pre-dose; 0.5 and 1 hourpost-dose (prior to second dose); and 1.5, 2, 2.5, 3, 4, 6, 8, 12, 18,24, 36, and 48 hours post-dose. Subjects were confined until 48 hourspost-dose and then returned 72 and 96 hours after the first dose foradditional blood sampling on an outpatient basis. Plasma concentrationsof colchicine and its metabolites were determined using validatedLC/MS-MS methods.

2-DMC concentrations were below the LOQ for all subjects. Eight of 13subjects had at least one measurable 3-DMC concentration (29 total 3-DMCmeasurable concentrations). 3-DMC concentrations ranged from 0.20 ng/mL(near the LOQ) to 0.45 ng/mL and were observed 1 to 4 hours post-dose.Given these low levels, metabolites are not discussed further herein.

When colchicine was administered in this low-dose regimen,concentrations increased to a maximum of 6.2 ng/mL, occurring 1.81 hoursafter the initial dose (0.81 hours after the second dose). Most of thesubjects (10 of 13 subjects or 77%) experienced a secondary peak within6 hours after the first of the two doses, attributed to intestinalsecretion and re-absorption and/or biliary recirculation.

The terminal elimination half-life was 23.6 hours. A summary of thepharmacokinetic parameter values is provided in in the table below.

TABLE 12A Colchicine Pharmacokinetic Parameter Values after Low-DoseColchicine (1.8 mg over 2 hours) Administration in Healthy AdultsC_(max) T_(max) Total AUC_(0-t) Total AUC_(∞) K_(el) CL/F V_(area)/Ft_(1/2) (pg/mL) (hr) (pg-hr/mL) (pg-hr/mL) (1/hr) (mL/hr) (L) (hr) N 1313 13 13 13 13 13 13 MEAN 6192.77 1.81 43787.55 52070.06 0.0326 36950.931188.72 23.63 STDEV 2433.70 0.38 11437.48 13689.27 0.0100 9993.17 319.569.24 % CV 39.30 21.24 26.12 26.29 30.80 27.04 26.88 39.10 MEDIAN 5684.002.00 43942.15 50783.77 0.0322 35444.40 1149.35 21.56 MIN 3160.00 1.0028821.45 34171.00 0.0141 24295.73 774.19 13.80 MAX 11370.00 2.5058931.99 74087.08 0.0502 52676.24 1724.36 49.20

Example 6 Pharmacokinetic Study in Healthy Adults of a Standard-DoseAcute Gout Regimen: 4.8 Mg Colchicine Over 6 Hours

This study was a single center, randomized, double-blind, double-dummypharmacokinetic and exploratory ECG safety study.

With respect to the pharmacokinetic aspect of the study, on Day 1,following a minimum of 10 hours overnight fast, subjects received theappropriate randomized study drug (combination of over-encapsulatedactive drug or placebo capsules such that the blind was preserved).Those randomized to colchicine received 4.8 mg over 6 hours (initially2×0.6 mg tablets followed by 1×0.6 mg tablet every hour for sixadditional doses). Those randomized to moxifloxacin received 1×400 mgtablet. Both dosing regimens were followed by a 4-hour post-dose fast(post-dose fast for colchicine arm started after the first dose ofcolchicine administered). Blood samples were obtained on Day 1 at thefollowing time points (relative to the first dose of study drug):pre-dose and 1 (prior to second dose), 3 (prior to fourth dose), 6(prior to final dose), 6.17, 6.33, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 10,12, 23, 36, 48, 72 and 96 hours post-dose. Subjects were confined until48 hours post-dose and then returned 72 and 96 hours after the firstdose for additional blood sampling on an outpatient basis. Plasmaconcentrations of colchicine and its metabolites were determined usingvalidated LC/MS-MS methods.

Eighteen healthy, non-smoking subjects with a mean age of 28.7 years(range 18 to 50 years) and within 15% of ideal body weight were enrolledin this study. Fifteen subjects were randomized to receive colchicineand 3 subjects were randomized to receive moxifloxacin as a positivecontrol for QTc prolongation. All subjects completed the study accordingto protocol.

When colchicine was administered as the standard-dose regimen used inthe treatment of patients experiencing an acute gout flare, colchicineconcentrations increased to a maximum of 6.8 ng/mL (similar to thereported Cmax in the low-dose regimen) and absorbed approximately 4.5hours after the initial dose (3.5 hours after the second dose). Most ofthe subjects (13 of 15 subjects receiving colchicine or 87%) experienceda secondary peak within 6 hours after a single oral dose, attributed tointestinal secretion and re-absorption and/or biliary recirculation. Theterminal elimination half-life was 31.38 hours. A summary of thepharmacokinetic parameter values is provided in the table below.

TABLE 13 MEAN (% CV) COLCHICINE PHARMACOKINETIC PARAMETER VALUES AFTERSTANDARD-DOSE COLCHICINE (4.8 MG OVER 6 HOURS) ADMINISTRATION IN HEALTHYADULTS C_(max) T_(max) Total AUC_(0-t) Total AUC_(∞) K_(el) CL/FV_(area)/F t_(1/2) (ng/mL) (hr) (ng-hr/mL) (ng-hr/mL) (h⁻¹) (mL/hr) (L)(hr) N 15 15 15 15 15 15 15 15 MEAN 6.84 4.47 104.95 118.20 0.024243168.87 1876.09 31.38 STDEV 1.30 1.99 24.61 26.01 0.0088 12862.03456.19 8.36 % CV 18.94 44.65 23.45 22.01 36.59 29.79 24.32 26.65 MEDIAN6.69 3.12 113.12 126.47 0.0212 37954.71 1902.14 32.76 MIN 4.95 3.1253.74 61.31 0.0147 31386.01 805.92 15.03 MAX 8.60 7.50 138.24 152.930.0461 78287.41 2639.21 47.22

2-DMC concentrations were below LOQ for all subjects. Fourteen of 15subjects had at least one measurable 3-DMC concentration; the 3-DMCconcentrations ranged from 0.25 ng/mL (near the LOQ) to 0.42 ng/mL andwere observed 1.12 to 12.12 hours post-dose. Summary mean 3-DMCpharmacokinetic parameter values can be found in the table below. Theobserved mean 3-DMC Cmax, AUC0-t, and AUC∞ concentrations wereapproximately 4.7%, 2%, and 4.1% of the observed mean colchicine Cmax,AUC0-t, AUC∞ concentrations, respectively.

TABLE 14 Mean (% CV) 3-DMC Pharmacokinetic Parameter Values afterStandard-Dose Colchicine (4.8 mg over 6 hours) Administration in HealthyAdults C_(max) T_(max) ¹ AUC_(0-t) AUC_(∞) Ke t_(1/2) (ng/mL) (h) (ng ·h/mL) (ng · h/mL) (h⁻¹) (h) N = 15 N = 14 N = 13 N = 8 N = 8 N = 8Standard Dose 0.32 5.06 2.09 4.84 0.1418 6.93 N = 15 (16.35) (3.12-8.12)(40.29) (42.73) (60.15) (64.35) ¹T_(max) reported mean (range)

Example 7 Food Effect Study Single Dose Vs. COL-PROBENECID® (0.5 MGCOLCHICINE/500 MG PROBENECID)

The clinical study of this example was a randomized, single-dose,three-way crossover study testing the bioequivalence of two formulationsof colchicine administered under standard fasting conditions, one the0.6 mgA colchicine formulation of Example 2 (test product) and one amarketed combination product, 0.5 mg colchicine/500 mg probenecidtablets (COL-PROBENECID®, Watson Laboratories, Inc.) (referenceproduct), and the effect of food on the test product by dosing followinga high-fat breakfast.

Twenty-eight healthy non-smoking adult volunteers (male and female) andno alternates initiated the study. Subjects received three single doses,in a randomized sequence of three treatment periods. On each occasion,subjects received either one colchicine tablet USP, 0.6 mg (testproduct) given either with food or in a standard fasting condition orone colchicine 0.5 mg/probenecid 500 mg tablet (reference product) givenin a standard fasting condition. Each treatment period was separated bya 14-day washout.

The two dosing conditions were (1) Standard Fasting Conditions (eithercolchicine tablets USP, 0.6 mg (test A) or reference product,COL-PROBENECID®): 1 tablet of test product (Test A) or reference productwith 240 mL of room temperature water after an overnight fast of atleast 10 hours; subjects will continue to fast for 4 hours post-dose;and (2) High-fat Breakfast (colchicine tablets USP, 0.6 mg): 1 tablet oftest product (Test B) with 240 mL of room temperature water 30 minutesafter initiation of a standardized, high-fat and high-calorie breakfast(FDA standard meal) preceded by an overnight fast.

Subjects were confined for at least 15 hours prior to and until at least24 hours after dosing each period. During each period, subjects returnedon four separate occasions for outpatient blood sampling.

No fluid, except that given with drug administration and thestandardized high-fat and high-calorie breakfast (FDA standard meal)depending on the randomization, was allowed from 1 hour prior to doseadministration until 2 hours after dosing. When fluids were restricted,they will be allowed ad libitum but will generally be controlled.

Dinner was served approximately 13.5 hours prior to dose administration.At 30 minutes before dose administration, those subjects to be dosedafter eating (depending on randomization) were served the standardized,high-fat and high-calorie breakfast (FDA standard meal). All subjectsfasted for at least 4 hours after dosing. Clear fluids, such as water,were allowed during fasting.

Subjects were served standardized meals and beverages, controlled by theclinic during periods of confinement. Meals were the same in content andquantity during each confinement period. No grapefruit and/or grapefruitcontaining products or caffeine and/or xanthine containing products wereallowed during the confinement portions of the study.

During confinement, only non-strenuous activity was permitted. Followingdose administration, subjects remained in a seated or upright positionfor at least 4 hours to ensure proper gastric emptying and subjectsafety.

Blood (6 mL) was collected in K2 EDTA vacutainers with samples takenwithin 1 hour prior to dosing (0 hour) and after dose administration at0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours (while confined) and 36,48, 72, and 96 hours (on an outpatient basis). 1. Colchicine andmetabolite plasma concentrations (colchicine, 2-DMC, 3-DMC, and 10demethylcolchicine) were measured using a validated bioanalyticalmethod.

Pharmacokinetic results comparing the test product under fed and fastingconditions are shown below.

TABLE 15 Pharmacokinetic results of colchicine test product under fedand fasting Ln-Transformed Data Least Squares Mean Geometric Mean 90%Confidence Interval PK Variable Test B Test A Test B Test A % Ratio(Lower Limit, Upper Limit) C_(max) (ng/mL) 7.784 7.781 2402.55 2393.60100.37  (89.84, 112.14) AUC_(0-t) (ng/mL-hr) 9.201 9.334 9906.4011310.90 87.58 (78.07, 98.26) AUC_(0-inf) (ng/mL-hr) 9.300 9.46810939.73 12939.64 84.54 (76.73, 93.15) Geometric means are based onleast squares means of ln-transformed values. Non-Transformed Data LeastSquares Mean 90% Confidence Interval PK Variable Test B Test A % Ratio(Lower Limit, Upper Limit) C_(max) (pg/mL) 2486.99 2493.15 99.75 (90.43, 109.07) AUC_(0-t) (pg/mL-hr) 10438.89 12536.56 83.27 (72.79,93.74) AUC_(0-inf) (pg/mL-hr) 11345.62 13907.83 81.58 (71.53, 91.63)T_(max) (hr) 1.85 1.35 137.14 (111.11, 163.17) Kel (hr⁻¹) 0.1902 0.1520125.13 (107.67, 142.58) T_(1/2) (hr) 4.34 6.27 69.17 (45.2, 93.14)

TABLE 16 Descriptive statistics for Pharmacokinetic Parameters for TestProduct A (0.6 mg) - Fasting conditions AUC0-t AUC0-inf Cmax (pg-hr/mL)(pg-hr/mL) (pg/mL) N 25 24 25 Arithmetic Mean 12589 14113 2503 STDev6210.729 5595.398 722.049 % CV 48.621 39.648 28.847 Median 11412.8012756.02 2473.00 Min 4430.73 6674.96 1291.00 Max 30787.30 27789.513989.00

TABLE 17 Descriptive statistics for Pharmacokinetic Parameters for TestProduct A (0.6 mg) - Fed conditions AUC0-t AUC0-inf Cmax (pg-hr/mL)(pg-hr/mL) (pg/mL) N 25 22 25 Arithmetic Mean 10491 11404 2497 STDev4024.804 2895.681 695.091 % CV 38.374 25.392 27.838 Median 9556.2510964.17 2293.00 Min 6168.53 7128.50 1256.00 Max 26031.15 20101.333930.00

Food was observed to have negligible effect on rate of absorption, asindicated by the percent ratio of ln-transformed Cmax data of 100.37,but decreased the extent of absorption by about 15%, as indicated by thepercent ratio of ln-transformed AUC0-t and AUC0-inf values of 87.56 and84.54, respectively. Under fasted and fed conditions, the mean Cmax was2.5 ng/mL. Tmax was 1.35 hrs under fasted conditions and 1.85 hrs underfed conditions.

Pharmacokinetic results comparing the test product to the referenceproduct are shown in the tables below. The difference in colchicinepotency of the test and reference products was corrected by calculatingdose-normalized values in the pharmacokinetic parameters.

TABLE 18 Summary of Statistical Analysis Colchicine Test Product A (0.6mg) - Fasting vs Reference Product C (0.5 mg) - Fasting (Dose Normalizedto 0.5 mg) N = 25 Ln-Transformed Data Least Squares Mean Geometric Mean90% Confidence Interval PK Variable Test A Reference C Test A ReferenceC % Ratio (Lower Limit, Upper Limit) C_(max) (ng/mL) 7.598 7.374 1994.671594.51 125.10 (111.97, 139.76) AUC_(0-t) (ng/mL-hr) 9.151 8.833 9425.756858.61 137.43  (122.5, 154.18) AUC_(0-inf) (ng/mL-hr) 9.286 8.97010783.03 7863.34 137.13 (124.46, 151.09) Geometric means are based onleast squares means of ln-transformed values. Non-Transformed Data LeastSquares Mean 90% Confidence Interval PK Variable Test A Reference C %Ratio (Lower Limit, Upper Limit) C_(max) (pg/mL) 2076.08 1688.54 122.95(110.07, 135.83) AUC_(0-t) (pg/mL-hr) 10435.91 8016.44 130.18 (115.25,145.11) AUC_(0-inf) (pg/mL-hr) 11565.28 8230.68 140.51 (126.04, 154.99)T_(max) (hr) 1.35 1.34 100.11  (74.05, 126.17) Kel (hr⁻¹) 0.1520 0.197077.16 (63.69, 90.63) T_(1/2) (hr) 6.27 3.78 165.89 (126.13, 205.65)

The 0.6 mgA colchicine tablet, formulated as in Example 2, showedenhanced bioavailability over COL-PROBENECID®. The formulation disclosedherein showed a greater rate and extent of absorption than did theCOL-PROBENECID®.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The endpoints of all ranges directed to thesame component or property are inclusive and independently combinable.

All methods described herein can be performed in a suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention as used herein. Unless defined otherwise, technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of skill in the art to which this invention belongs.The terms wt %, weight percent, percent by weight, etc. are equivalentand interchangeable

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those preferred embodiments may become apparent to those of ordinaryskill in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

We claim:
 1. A colchicine dosage form, comprising colchicine; and a pharmaceutically acceptable excipient; wherein the colchicine dosage form comprises no more than 0.42% N-deacetyl-N-formyl colchicine.
 2. The dosage form of claim 1, wherein the colchicine dosage form further comprises no more than 2.0% of Impurity B.
 3. The dosage form of claim 2, wherein the colchicine dosage form further comprises no more than 1.5% of Impurity B.
 4. The dosage form of claim 1, wherein the pharmaceutically acceptable excipient is a filler, a binder, a disintegrant, a lubricant, or a combination comprising at least one of the foregoing excipients.
 5. The dosage form of claim 4, wherein the filler is microcrystalline cellulose, starch, lactose, sucrose, glucose, mannitol, dextrose, silicic acid, or a combination comprising at least one of the foregoing fillers.
 6. The dosage form of claim 5, wherein the filler is present in an amount of about 10 wt % to about 99 wt %, based on the total weight of the dosage form.
 7. The dosage form of claim 4, wherein the binder is pregelatinized starch, carboxymethylcellulose, alignate, gelatin, polyvinylpyrrolidone, acacia, or a combination comprising at least one of the foregoing binders.
 8. The dosage form of claim 7, wherein the binder is present in an amount of about 10 wt % to about 99 wt %, based on the total weight of the dosage form.
 9. The dosage form of claim 4, wherein the disintegrant is sodium starch glycolate, sodium croscarmellose (cross-linked carboxy methyl cellulose), crosslinked polyvinylpyrrolidone (PVP-XL), anhydrous calcium hydrogen phosphate, agar-agar, potato or tapioca starch, alginic acid, or a combination comprising at least one of the foregoing disintegrants.
 10. The dosage form of claim 9, wherein the disintegrant is present in an amount of about 1 to about 20 wt %, based on the total weight of the dosage form.
 11. The colchicine dosage form of claim 1, wherein the colchicine is present in an amount of about 0.6 mg.
 12. The colchicine dosage form of claim 1 compressed into a tablet.
 13. The colchicine dosage form of claim 1, wherein the percent N-deacetyl-N-formyl colchicine in the colchicine dosage form is determined in an HPLC assay under the following conditions: the mobile phase is pH 4.5 Ammonium Acetate Buffer:methanol gradient; the column is a Waters XBridge C18, 250 mm×4.6 mm, 5 μm particle size; the flow rate is 0.9 mL/min; the column temperature is 10±3.5 C and the sample temperature is 10±2 C; the detection is at 246 nm; the injection volume is 75 μL; and the run time is 60 minutes.
 14. A method for treating a patient in need of colchicine therapy, wherein the method comprises administering to a patient in need of colchicine therapy a therapeutically effective amount of the colchicine dosage form of claim
 1. 15. The method of claim 14, wherein administering comprises orally administering 1.2 mg colchicine to a human patient at onset of a gout flare; and then orally administering 0.6 mg colchicine to the patient about one hour after the first administration.
 16. A method of determining response of patients having a gout flare to colchicine therapy, comprising administering a dosing regimen to a patient having a gout flare; and determining likelihood of percent improvement in pain of the patient at 24 hours after the first dose; wherein the dosing regimen is a first dosing regimen consisting of two colchicine tablets at the onset of the gout flare, followed by one colchicine tablet in about one hour or a second dosing regimen consisting of two colchicine tablets at the onset of the gout flare, followed by one colchicine tablet every hour for 6 hours, wherein a colchicine tablet comprises about 0.6 mg colchicine. 